Targeted binding agents directed to dll4 and uses thereof

ABSTRACT

The invention relates to targeted binding agents against DLL4 and uses of such agents. More specifically, the invention relates to fully human monoclonal antibodies directed to DLL4. The described targeted binding agents are useful in the treatment of diseases associated with the activity and/or overproduction of DLL4 and as diagnostics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/446,637, filed on Apr. 13, 2012, now U.S. Pat. No. 8,663,636, issuedon Mar. 4, 2014, said application Ser. No. 13/446,637 is a continuationof U.S. application Ser. No. 12/562,268, filed on Sep. 18, 2009, nowU.S. Pat. No. 8,192,738, issued on Jun. 5, 2012, said application Ser.No. 12/562,268 claims benefit under 35 U.S.C. §119(e) of the followingU.S. Provisional Patent Application No. 61/098,673 filed on Sep. 19,2008. Each of the above listed applications is incorporated by referenceherein in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled DLL4_(—)100US3_seqlistcreated on Jan. 14, 2014 and having a size of 80.0 kilobytes.

FIELD OF THE INVENTION

The invention relates to targeted binding agents against DLL4 and usesof such agents. More specifically, the invention relates to fully humanmonoclonal antibodies directed to DLL4. The described targeted bindingagents are useful in the treatment of diseases associated with theactivity and/or overproduction of DLL4 and as diagnostics.

DESCRIPTION OF THE RELATED ART

The Notch signaling cascade is an evolutionarily conserved pathway thathas been implicated in cell fate determination, stem cell maintenanceand differentiation in many tissues during development. Thus far, fourNotch receptor ligands (Notch1-4) and five ligands (Jagged-1/2 andDelta-like ligand (Dll) 1/3/4) have been identified in mammals. Notchreceptors exist as heterodimers, comprised of two non-covalentlyassociated extracellular and transmembrane subunits. Ligand binding tothe extracellular subunit triggers proteolytic cleavages by enzymes suchas TNFα converting enzyme (TACE) and gamma-secretase which results inthe creation of Notch intracellular domains (NICD), which translocate tothe nucleus and bind to transcription factors which ultimately resultsin the activation of downstream target genes (see Bray, 2006, Nat. Rev.Mol. Cell. Biol., 7, 678).

Emerging evidence suggests that multiple Notch pathway components areexpressed in the vasculature and that aberrations in normal Notchsignaling can result in vascular phenotypes. For example, mutations inJagged 1 and Notch 3 result in Alagille syndrome and cerebral autosomaldominant arteriopathy with subcortical infarcts and leukoencephalopathy,respectively, two disorders that exhibit vascular defects. Furthermore,genetic deletion of Notch1 and DLL4 in mice all result in embryoniclethality with vascular abnormalities. In addition, deletion of a singleallele of DLL4 in mice results in embryonic lethality with severevascular defects in most genetic backgrounds (Duarte et al., 2004, GenesDev., 18, 2474; Gale et al., 2004, Proc. Nat. Acad. Sci., 101, 15949).This phenotype has only previously been reported for VEGF-A and suggeststhat DLL4 may play an important role in vascular development.

DLL4 expression has also been reported on the vasculature of tumors fromhuman clear-cell renal cell carcinomas, glioblastomas and cancers of thebreast and bladder (Mailhos et al., 2001, Differentiation, 69, 135;Patel et al., 2005, Cancer Res., 65, 8690; Patel et al., 2006, Clin.Cancer Res., 12, 4836; Li et al., 2007, Cancer Res., 67, 11244). Arecent study has also suggested that DLL4 may be expressed on a smallproportion of tumor cells in human glioblastoma (Li et al., 2007, CancerRes., 67, 11244). The effect of blocking DLL4 signaling on tumor growthhas also been evaluated in several preclinical models of cancer in whichtumor cell lines are grown subcutaneously in immunodeficient mice(Ridgway et al., 2006, Nature, 444, 1083; Noguera-Troise et al., Nature,444, 1032). In these studies, reductions in tumor growth of have beenreported. These anti-tumor effects were associated with an increase inthe density of poorly functional vessels in the tumors concomitant withan increase in hypoxia. Taken together, these data suggest that, inaddition to its role in vascular development, DLL4 may also play a rolein development of the tumor vasculature.

In addition to effects on angiogenesis, Notch signaling has also beenimplicated in cancer stem cells from multiple tumor types (Dontu et al.,2004, Breast Can. Res., 6, R605; Wilson & Radtke, 2006, FEBS Lett., 580,2860). Cancer stem cells have been isolated from a variety ofheamtopoietic and solid tumors (Al-Hajj et al., 2003, Proc. Nat. Acad.Sci., 100, 3983; Lapidot et al., 1994, Nature, 17, 645; Tan et al.,2006, Laboratory Investigation, 86, 1203) and the presence of DLL4 onsmall populations of tumor cells further suggests that DLL4 may also beinvolved in cancer stem cell biology and that DLL4 antagonists maypartially mediate anti-tumor effects through interactions with thesecell types.

SUMMARY OF THE INVENTION

The present invention relates to targeted binding agents thatspecifically bind to DLL4 and inhibit the biological activity of DLL4.Embodiments of the invention relate to targeted binding agents thatspecifically bind to DLL4 and inhibit binding of DLL4 to a Notchreceptor (e.g., Notch 1, 2, 3 or 4).

Embodiments of the invention relate to targeted binding agents thatspecifically bind to DLL4 and inhibit binding of DLL4 to a Notchreceptor (e.g., Notch 1 or 4). In one embodiment of the invention thetargeted binding agent specifically binds to DLL4 and inhibits bindingto a Notch receptor, e.g., Notch 1. In one embodiment the targetedbinding agent inhibits at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% of DLL4 binding to a Notch receptor, (e.g., Notch 1), compared tobinding that would occur in the absence of the targeted binding agent.

In some embodiments of the invention, the targeted binding agent bindsDLL4 with a binding affinity (K_(D)) of less than 5 nanomolar (nM). Inother embodiments, the targeted binding agent binds with a K_(D) of lessthan 4 nM, 3 nM, 2 nM or 1 nM. In some embodiments of the invention, thetargeted binding agent binds DLL4 with a K_(D) of less than 950picomolar (pM). In some embodiments of the invention, the targetedbinding agent binds DLL4 with a K_(D) of less than 900 pM. In otherembodiments, the targeted binding agent binds with a K_(D) of less than800 pM, 700 pM or 600 pM. In some embodiments of the invention, thetargeted binding agent binds DLL4 with a K_(D) of less than 500 pM. Inother embodiments, the targeted binding agent binds with a K_(D) of lessthan 400 pM. In still other embodiments, the targeted binding agentbinds with a K_(D) of less than 300 pM. In some other embodiments, thetargeted binding agent binds with a K_(D) of less than 200 pM. In someother embodiments, the targeted binding agent binds with a K_(D) of lessthan 150 pM. In yet another embodiment, the targeted binding agent bindswith a K_(D) of less than 100 pM. In another embodiment, the targetedbinding agent binds with a K_(D) of less than 50 pM. In one specificembodiment, the targeted binding agent of the invention can bind humanDLL4 with an affinity K_(D) of less than 10 pM. In another specificembodiment, the targeted binding agent of the invention can bind humanDLL4 with an affinity K_(D) of less than 1 pM. The K_(D) may be assessedusing a method described herein or known to one of skill in the art(e.g., a BIAcore assay, ELISA, FACS) (Biacore International AB, Uppsala,Sweden).

The binding properties of the targeted binding agent or antibody of theinvention may also be measured by reference to the dissociation orassociation rates (k_(off) and k_(on) respectively).

In one embodiment of the invention, a targeted binding agent or anantibody may have an k_(on) rate (antibody (Ab)+antigen (Ag)^(k) ^(on)→Ab-Ag) of at least 10⁴ M⁻¹ s⁻¹, at least 5×10⁴ M⁻¹ s⁻¹, at least 10⁵M⁻¹ s⁻¹, at least 2×10⁵ M⁻¹ s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹, at least 10⁶M⁻¹ s⁻¹, at least 5×10⁶ M⁻¹ s⁻¹, at least 10⁷ M⁻¹ s⁻¹, at least 5×10⁷M⁻¹ s⁻¹, or at least 10⁸ M⁻¹ s⁻¹.

In another embodiment of the invention, targeted binding agent or anantibody may have a k_(off) rate ((Ab-Ag)^(k) ^(off) →antibody(Ab)+antigen (Ag)) of less than 5×10⁻¹ s⁻¹, less than 10⁻¹ s⁻¹, lessthan 5×10⁻² s⁻¹, less than 10⁻² s⁻¹, less than 5×10⁻³ s⁻¹, less than10⁻³ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 4×10⁻⁴ s⁻¹, less than 3×10⁻⁴s⁻¹, less than 2×10 s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁵ s⁻¹, lessthan 10⁻⁵ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁶ s⁻¹, less than5×10⁻⁷ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁸s⁻¹, less than 5×10⁻⁹ s⁻¹, less than 10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

The targeted binding agent of the invention binds human DLL4. In someexamples the targeted binding agent of the invention is cross-reactivewith other DLL4 proteins from other species. In one embodiment, thetargeted binding agent of the invention is cross-reactive withcynomolgus monkey DLL4. In another embodiment, the targeted bindingagent of the invention is cross-reactive with cynomolgus monkey DLL4 butis only weakly cross-reactive with DLL4 proteins from other species,e.g., is only weakly cross-reactive with mouse DLL4, e.g., the targetedbinding agent binds mouse DLL4 with a K_(D) of more than 360 nM asassessed by BIAcore technology.

In another embodiment, the targeted binding agent of the invention hasnearly equivalent affinity for DLL4 proteins from other species. In onespecific example, the human DLL4 targeted binding agent of the inventionhas nearly equivalent affinity for cynomolgus monkey DLL4. By equivalentlevel of affinity we mean that when the affinity with respect to humanDLL4 is 1, the affinity of the antibody with respect to cynomolgusmonkey DLL4 is between 0.2-5 or between 0.2-2.

In another embodiment, the targeted binding agent of the invention isspecific for human DLL4 but does not bind other Notch ligands. In oneexample, the targeted binding agent of the invention is specific forhuman DLL4 but does not bind significantly to DLL1, e.g., the DLL1/mockratio is less than 2.5 as determined by testing the ability of atargeted binding agent as described herein (15-300 μg/ml) to bind 293Tcells transiently transfected with human DLL1 or HEK293 cells stablytransfected with Dll1. In another example, the targeted binding agent ofthe invention is specific for human DLL4 but does not bind significantlyto Jagged 1, e.g., the DLL1/mock ratio is less than 1.5 as determined bytesting the ability of a targeted binding agent as described herein (5μg/ml) to bind 293T cells transiently transfected with human Jagged 1 orHEK293 cells stably transfected with human Jagged 1.

In yet another embodiment, the targeted binding agent of the inventioninhibits DLL4-Notch1 receptor-ligand binding. In one example, activitypossessed by the targeted binding agent can be demonstrated at an IC₅₀concentration (a concentration to achieve 50% inhibition of) below 10μM. In another example, the targeted binding agent of the invention canhave an IC₅₀ concentration of less than 50, 40, 30, 20, 10, 5, 4, 2, 1,0.8, 0.7, 0.6, 0.5 or 0.4 nM.

In yet another embodiment, the targeted binding agent of the inventioncan have both in vitro and in vivo activity. In one specific example,the targeted binding agent reverses DLL4-stimulated inhibition of HUVECcell proliferation in 2D culture. In one example, the antibodies of theinvention can reverse DLL4 stimulated inhibition of HUVEC cellproliferation by 70% or over, e.g., 75%, 80%, 85%, 90%, or 95% comparedto a control (using the assay of example 9 where no DLL4 is added).

In still yet another embodiment, the targeted binding agents, e.g.,antibodies, of the invention can inhibit HUVEC tube formation in 2Dculture. For example, the antibodies of the invention can exhibitgreater than 50% inhibition, e.g., 50%, 60%, 70%, 80%, 90%, or 95%inhibition at a concentration of 0.08 μg/ml relative to a control (thecontrol is the maximal inhibitory effect determined using 20 μg/ml ofthe same antibody).

In yet another embodiment, the targeted binding agents of the inventioncan exhibit less than 50%, e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%or 5 internalization at four hours relative to t=0 control. In anotherembodiment, the targeted binding agents of the invention can exhibitbetween 5-50%, 10-40% or 20-40% internalization at four hours relativeto t=0 control (see example 15).

In one embodiment, the targeted binding agents of the invention thatspecifically binds to DLL4 can exhibit one or more of the followingproperties, including binds human DLL4 with a K_(D) of less than 200 pM;cross-reacts with cynolmolgus monkey DLL4; weakly cross-reacts withmouse DLL4; binds cynomologus DLL4 with nearly equivalent affinity; doesnot bind significantly to DLL1 or Jagged 1; exhibits over 85% reverseDLL4-stimulated inhibition of HUVEC cell proliferation in 2D culturecompared to a control; exhibits greater than 50% inhibition of HUVECcell tube formation in 2D culture at a concentration of 0.08 μg/mlrelative to a control; and exhibits less than 50% internalizationrelative at four hours relative to t=0 control.

In another embodiment, the targeted binding proteins disclosed hereinpossess beneficial efficacious, metabolic, and/or pharmacodynamicproperties.

In another embodiment of the invention, the targeted binding agentcompetes with any one of fully human monoclonal antibodies of 4B4, 2H10,21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4 or 21H3RK forbinding to Notch 1.

In some embodiments of the invention, the targeted binding agentinhibits tumor growth and/or metastasis in a mammal. In anotherembodiment, the targeted binding agent can treat a condition associatedwith angiogenesis.

In some embodiments of the invention, the targeted binding agent is anantibody. In some embodiments of the invention, the targeted bindingagent is a monoclonal antibody. In one embodiment of the invention, thetargeted binding agent is a fully human monoclonal antibody. In anotherembodiment of the invention, the targeted binding agent is a fully humanmonoclonal antibody of the IgG1, IgG2, IgG3 or IgG4 isotype. In anotherembodiment of the invention, the targeted binding agent is a fully humanmonoclonal antibody of the IgG2 isotype. This isotype has reducedpotential to elicit effector function in comparison with other isotypes,which may lead to reduced toxicity. In another embodiment of theinvention, the targeted binding agent is a fully human monoclonalantibody of the IgG1 isotype. The IgG1 isotype has increased potentialto elicit ADCC in comparison with other isotypes, which may lead toimproved efficacy. The IgG1 isotype has improved stability in comparisonwith other isotypes, e.g. IgG4, which may lead to improvedbioavailability, or improved ease of manufacture or a longer half-life.In one embodiment, the fully human monoclonal antibody of the IgG1isotype is of the z, za or f allotype.

A further embodiment is a targeted binding agent or an antibody thatspecifically binds to DLL4 and comprises a sequence comprising one ofthe complementarity determining regions (CDR) sequences shown in Table2. Embodiments of the invention include a targeted binding agent orantibody comprising a sequence comprising: any one of a CDR1, a CDR2 ora CDR3 sequence as shown in Table 2. A further embodiment is a targetedbinding agent or an antibody that specifically binds to DLL4 andcomprises a sequence comprising two of the CDR sequences shown in Table2. In another embodiment the targeted binding agent or antibodycomprises a sequence comprising a CDR1, a CDR2 and a CDR3 sequence asshown in Table 2. In another embodiment the targeted binding agent orantibody comprises a sequence comprising one of the CDR sequences shownin Table 2. Embodiments of the invention include a targeted bindingagent or antibody comprising a sequence comprising: any one of a CDR1, aCDR2 or a CDR3 sequence as shown in Table 2. In another embodiment thetargeted binding agent or antibody comprises a sequence comprising twoof the CDR sequences shown in Table 2. In another embodiment thetargeted binding agent or antibody comprises a sequence comprising aCDR1, a CDR2 and a CDR3 sequence as shown in Table 2. In anotherembodiment the targeted binding agent or antibody may comprise asequence comprising a CDR1, a CDR2 and a CDR3 sequence of a variableheavy chain sequence or a variable light chain sequence as shown inTable 2. In some embodiments, the targeted binding agent is an antibody.In certain embodiments, the targeted binding agent is a fully humanmonoclonal antibody. In certain other embodiments, the targeted bindingagent is a binding fragment of a fully human monoclonal antibody.

In another embodiment, the targeted binding agent comprises a VH CDR1 asshown in Table 2, wherein the sequence of the VH CDR1 has an amino acidsequence identical to or comprising 1, 2, or 3 amino acid residuesubstitutions relative to the VH CDR1 as shown in Table 2; a VH CDR2 asshown in Table 2 having an amino acid sequence identical to orcomprising 1, 2, or 3 amino acid residue substitutions relative to theVH CDR2 as shown in Table 2; a VH CDR3 as shown in Table 2 having anamino acid sequence identical to or comprising 1, 2, or 3 amino acidresidue substitutions relative to the VH CDR3 as shown in Table 2; a VLCDR1 as shown in Table 2 having an amino acid sequence identical to orcomprising 1, 2, or 3 amino acid residue substitutions relative to VLCDR1 as shown in Table 2; a VL CDR2 as shown in Table 2 having an aminoacid sequence identical to or comprising 1, 2, or 3 amino acid residuesubstitutions relative to the VL CDR2 as shown in Table 2; and a VL CDR3as shown in Table 2 having an amino acid sequence identical to orcomprising 1, 2, or 3 amino acid residue substitutions relative to theVL CDR3 as shown in Table 2.

In one embodiment, the targeted binding agent comprises a VH CDR1, CDR2and CDR3 as shown in Table 2 and a VL CDR1 CDR2 and CDR3 as shown inTable 2.

In yet another embodiment, the targeted binding agent immunospecificallybinds DLL4 and comprises a heavy chain variable domain as shown in table2 having at least 90% identity to the amino acid as shown in table 2 andcomprises a light chain variable domain as shown in table 2 having atleast 90% identity to the amino acid sequence as shown in table 2,wherein said antibody has the activity of binding to DLL4.

In another embodiment the targeted binding agent may comprise a sequencecomprising any one of the CDR1, CDR2 or CDR3 of the variable heavy chainsequences encoded by a polynucleotide in a plasmid designatedMab2H10VHOP, Mab9G8VH, Mab21H3VH, and Mab4B4VH which were deposited atthe American Type Culture Collection (ATCC) under number PTA-9502,PTA-9517, PTA-9501, or PTA-9508 on Sep. 17, 2008. In another embodimentthe targeted binding agent may comprise a sequence comprising any one ofthe CDR1, CDR2 or CDR3 of the variable light chain sequences encoded bya polynucleotide in a plasmid designated Mab9G8VLOPT1, Mab21H3VLOP, andMab4B4VL which were deposited at the American Type Culture Collection(ATCC) under number PTA-9516, PTA-9500 or PTA-9520 on Sep. 17, 2008 andMab2H10VLOP which was deposited on Jul. 7, 2009 under number PTA-10181.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab2H10VHOP which was deposited at the American Type Culture Collection(ATCC) under number PTA-9502 on Sep. 17, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab2H10VHOP which was deposited at the American Type Culture Collection(ATCC) under number PTA-9502 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising a CDR3 encoded by the polynucleotide inplasmid designated Mab2H10VLOP which was deposited at the American TypeCulture Collection (ATCC) under number PTA-10181 on Jul. 7, 2009.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab2H10VHOP which was deposited at the American Type Culture Collection(ATCC) under number PTA-9502 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab2H10VLOP which was deposited at the American Type Culture Collection(ATCC) under number PTA-10181 on Jul. 7, 2009.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab2H10VHOP which was deposited at the American Type Culture Collection(ATCC) under number PTA-9502 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising at least one, at least two, or at leastthree of the CDRs of the antibody encoded by the polynucleotide inplasmid designated Mab2H10VLOP which was deposited at the American TypeCulture Collection (ATCC) under number PTA-10181 on Jul. 7, 2009.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab9G8VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9517.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab9G8VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9517 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising a CDR3 encoded by the polynucleotide inplasmid designated Mab9G8VLOPT1 which was deposited at the American TypeCulture Collection (ATCC) under number PTA-9516 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab9G8VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9517 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab9G8VLOPT1 which was deposited at the American Type Culture Collection(ATCC) under number PTA-9516 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab9G8VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9517 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising at least one, at least two, or at leastthree of the CDRs of the antibody encoded by the polynucleotide inplasmid designated Mab9G8VLOPT1 which was deposited at the American TypeCulture Collection (ATCC) under number PTA-9516 on Sep. 17, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab21H3VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9501 on Sep. 17, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab21H3VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9501 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising a CDR3 encoded by the polynucleotide inplasmid designated Mab21H3VLOP which was deposited at the American TypeCulture Collection (ATCC) under number PTA-9500 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab21H3VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9501 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab21H3VLOP which was deposited at the American Type Culture Collection(ATCC) under number PTA-9500 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab21H3VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9501 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising at least one, at least two, or at leastthree of the CDRs of the antibody encoded by the polynucleotide inplasmid designated Mab21H3VLOP which was deposited at the American TypeCulture Collection (ATCC) under number PTA-9500 on Sep. 17, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab4B4VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9508 on Sep. 17, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designatedMab4B4VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9508 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising a CDR3 encoded by the polynucleotide inplasmid designated Mab4B4VL which was deposited at the American TypeCulture Collection (ATCC) under number PTA-9520 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid esignated Mab4B4VHwhich was deposited at the American Type Culture Collection (ATCC) undernumber PTA-9508 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab4B4VL which was deposited at the American Type Culture Collection(ATCC) under number PTA-9520 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designatedMab4B4VH which was deposited at the American Type Culture Collection(ATCC) under number PTA-9508 on Sep. 17, 2008 and a variable light chainamino acid sequence comprising at least one, at least two, or at leastthree of the CDRs of the antibody encoded by the polynucleotide inplasmid designated Mab4B4VL which was deposited at the American TypeCulture Collection (ATCC) under number PTA-9520 on Sep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab2H10VHOP which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9502 onSep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab9G8VH which was deposited at theAmerican Type Culture Collection (ATCC) under number PTA-9517 on Sep.17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab21H3VH which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9501 onSep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab4B4VH which was deposited at theAmerican Type Culture Collection (ATCC) under number PTA-9508 on Sep.17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab2H10VLOP which was deposited atthe American Type Culture Collection (ATCC) under number PTA-10181 onJul. 7, 2009.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab9G8VLOPT1 which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9516 onSep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab21H3VLOP which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9500 onSep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab4B4VL which was deposited at theAmerican Type Culture Collection (ATCC) under number PTA-9520 on Sep.17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab2H10VHOP which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9502 onSep. 17, 2008 and a a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab2H10VLOP which was deposited atthe American Type Culture Collection (ATCC) under number PTA-10181 onJul. 7, 2009.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab9G8VLOPT1 which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9516 onSep. 17, 2008 and a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab9G8VH which was deposited at theAmerican Type Culture Collection (ATCC) under number PTA-9517 on Sep.17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable heavy chain of an antibody encoded by thepolynucleotide in plasmid designated Mab21H3VH which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9501 onSep. 17, 2008 and a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab21H3VLOP which was deposited atthe American Type Culture Collection (ATCC) under number PTA-9500 onSep. 17, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab4B4VL which was deposited at theAmerican Type Culture Collection (ATCC) under number PTA-9520 on Sep.17, 2008 and a a variable light chain of an antibody encoded by thepolynucleotide in plasmid designated Mab4B4VL which was deposited at theAmerican Type Culture Collection (ATCC) under number PTA-9520 on Sep.17, 2008.

It is noted that those of ordinary skill in the art can readilyaccomplish CDR determinations. See for example, Kabat et al., Sequencesof Proteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3. Kabat provides multiplesequence alignments of immunoglobulin chains from numerous speciesantibody isotypes. The aligned sequences are numbered according to asingle numbering system, the Kabat numbering system. The Kabat sequenceshave been updated since the 1991 publication and are available as anelectronic sequence database (latest downloadable version 1997). Anyimmunoglobulin sequence can be numbered according to Kabat by performingan alignment with the Kabat reference sequence. Accordingly, the Kabatnumbering system provides a uniform system for numbering immunoglobulinchains.

In one embodiment, the targeted binding agent or antibody comprises asequence comprising any one of the heavy chain sequences shown in Table2. In another embodiment, the targeted binding agent or antibodycomprises a sequence comprising any one of the heavy chain sequences ofantibodies 20G8, 21H3, 14B1, 18B7, 17B6, 17F3, 12A1, 17G12, 19G9, 21F7,20D6, 1D4, 4B4, 2H10, or 21H3RK, or any optimized version of the heavychain sequences of these antibodies as shown in Table 5, 7, 9, 11 or 13.Light-chain promiscuity is well established in the art, thus, a targetedbinding agent or antibody comprising a sequence comprising any one ofthe light chain sequences of antibodies 4B4, 2H10, 21F7, 12A1, 17F3,9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK, or any optimized versionof the light chain sequences of these antibodies as shown in Table 6, 8,10, 12 or 13, or any other antibody as disclosed herein. In oneembodiment, the targeted binding agent or antibody comprises a sequencecomprising any one of the heavy chain sequences shown in Table 2, or anyoptimized version of the heavy chain sequences of these antibodies asshown in Table 5, 7, 9, 11 or 13, and may further comprise any one ofthe light chain sequences shown in Table 6, 8, 10, 12 or 13 or anotherantibody as disclosed herein. In some embodiments, the antibody is afully human monoclonal antibody.

In some embodiments, the targeting binding agent is a monoclonalantibody selected from the group consisting of: 4B4, 2H10, 21F7, 12A1,17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. In one embodiment,the targeted binding agent comprises one or more of fully humanmonoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4,3A7, 4B3, 1D4, or 21H3RK. In certain embodiments, the targeting bindingagent is monoclonal antibody 4B4. In certain embodiments, the targetingbinding agent is monoclonal antibody 21H3. In certain embodiments, thetargeting binding agent is monoclonal antibody 2H10. In certainembodiments, the targeting binding agent is monoclonal antibody 9G8. Incertain other embodiments, the targeting binding agent is monoclonalantibody 21H3RK.

In one embodiment a targeted binding agent or an antibody may comprise asequence comprising a heavy chain CDR1, CDR2 and CDR3 selected from anyone of the sequences shown in Table 2

In one embodiment a targeted binding agent or an antibody may comprise asequence comprising a light chain CDR1, CDR2 and CDR3 selected from anyone of the sequences shown in Table 2. In one embodiment a targetedbinding agent or an antibody may comprise a sequence comprising a heavychain CDR1, CDR2 and CDR3 selected from any one of the CDRs ofantibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3,1D4, or 21H3RK. In one embodiment a targeted binding agent or anantibody may comprise a sequence comprising a light chain CDR1, CDR2 andCDR3 selected from any one of the CDRs of antibodies 4B4, 2H10, 21F7,12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

In another embodiment the targeted binding agent or antibody maycomprise a sequence comprising any one of a CDR1, a CDR2 or a CDR3 of avariable heavy chain sequence of any one of the fully human monoclonalantibodies 4B4 or 21H3, 2H10, 9G8, or 21H3RK, as shown in Table 2. Inanother embodiment the targeted binding agent or antibody may comprise asequence comprising any one of a CDR1, a CDR2 or a CDR3 of a variablelight chain sequence of any one of the fully human monoclonal antibodies4B4 or 21H3, 2H10, 9G8, or 21H3RK, as shown in Table 2. In anotherembodiment the targeted binding agent or antibody may comprise asequence comprising a CDR1, a CDR2 and a CDR3 of fully human monoclonalantibody 4B4 or 21H3, 2H10, 9G8, or 21H3RK, as shown in Table 2, and aCDR1, a CDR2 and a CDR3 sequence of fully human monoclonal antibody 4B4or 21H3, 2H10, 9G8, or 21H3RK, as shown in Table 2. In some embodiments,the antibody is a fully human monoclonal antibody.

In another embodiment the targeted binding agent or antibody comprises asequence comprising the CDR1, CDR2 and CDR3 sequence of fully humanmonoclonal antibody 4B4 as shown in Table 2 and the CDR1, CDR2 and CDR3sequence of fully human monoclonal antibody 4B4 as shown in Table 2. Inanother embodiment the targeted binding agent or antibody comprises asequence comprising the CDR1, CDR2 and CDR3 sequence of fully humanmonoclonal antibody 21H3 as shown in Table 2 and the CDR1, CDR2 and CDR3sequence of fully human monoclonal antibody 21H3 as shown in Table 2. Inanother embodiment the targeted binding agent or antibody comprises asequence comprising the CDR1, CDR2 and CDR3 sequence of fully humanmonoclonal antibody 2H10 as shown in Table 2 and the CDR1, CDR2 and CDR3sequence of fully human monoclonal antibody 2H10 as shown in Table 2. Insome embodiments, the antibody is a fully human monoclonal antibody.

A further embodiment of the invention is a targeted binding agent orantibody comprising a sequence comprising the contiguous sequencespanning the framework regions and CDRs, specifically from FR1 throughFR4 or CDR1 through CDR3, of any one of the sequences as shown in Table2 or Table 13. In one embodiment the targeted binding agent or antibodycomprises a sequence comprising the contiguous sequences spanning theframework regions and CDRs, specifically from FR1 through FR4 or CDR1through CDR3, of any one of the sequences of monoclonal antibodies 4B4or 21H3, 2H10, 9G8, or 21H3RK, as shown in Table 2 or Table 13. In someembodiments, the antibody is a fully human monoclonal antibody.

In another embodiment the agent or antibody, or antigen-binding portionthereof, comprises a heavy chain polypeptide comprising the sequence ofSEQ ID NO.: 2. In one embodiment, the agent or antibody, orantigen-binding portion thereof, further comprises a light chainpolypeptide comprising the sequence of SEQ ID NO.: 4. In someembodiments, the antibody is a fully human monoclonal antibody.

One embodiment provides a targeted binding agent or antibody, orantigen-binding portion thereof, wherein the agent or antibody, orantigen-binding portion thereof, comprises a heavy chain polypeptidecomprising the sequence of SEQ ID NO.: 6. In one embodiment, the agentor antibody, or antigen-binding portion thereof, further comprises alight chain polypeptide comprising the sequence of SEQ ID NO.: 8. Insome embodiments, the antibody is a fully human monoclonal antibody.

In another embodiment the agent or antibody, or antigen-binding portionthereof, comprises a heavy chain polypeptide comprising the sequence ofSEQ ID NO.: 22. In another embodiment, the agent or antibody, orantigen-binding portion thereof, further comprises a light chainpolypeptide comprising the sequence of SEQ ID NO.: 24. In someembodiments, the antibody is a fully human monoclonal antibody.

In another embodiment the agent or antibody, or antigen-binding portionthereof, comprises a heavy chain polypeptide comprising the sequence ofSEQ ID NO.: 30. In another embodiment, the agent or antibody, orantigen-binding portion thereof, further comprises a light chainpolypeptide comprising the sequence of SEQ ID NO.: 32. In someembodiments, the antibody is a fully human monoclonal antibody.

In another embodiment the agent or antibody, or antigen-binding portionthereof, comprises a heavy chain polypeptide comprising the sequence ofSEQ ID NO.: 30. In another embodiment, the agent or antibody, orantigen-binding portion thereof, further comprises a light chainpolypeptide comprising the sequence of SEQ ID NO.: 50. In someembodiments, the antibody is a fully human monoclonal antibody.

In one embodiment the targeted binding agent or antibody comprises asmany as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, fouror five, amino acid additions, substitutions, deletions, and/orinsertions within the disclosed CDRs or heavy or light chain sequences.Such modifications may potentially be made at any residue within theCDRs. In some embodiments, the antibody is a fully human monoclonalantibody.

In one embodiment, the targeted binding agent or antibody comprisesvariants or derivatives of the CDRs disclosed herein, the contiguoussequences spanning the framework regions and CDRs (specifically from FR1through FR4 or CDR1 through CDR3), the light or heavy chain sequencesdisclosed herein, or the antibodies disclosed herein. Variants includetargeted binding agents or antibodies comprising sequences which have asmany as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, four,five or six amino acid additions, substitutions, deletions, and/orinsertions in any of the CDR1, CDR2 or CDR3s as shown in Table 2 orTable 13, the contiguous sequences spanning the framework regions andCDRs (specifically from FR1 through FR4 or CDR1 through CDR3) as shownin Table 2 or Table 13, the light or heavy chain sequences disclosedherein, or with the monoclonal antibodies disclosed herein. Variantsinclude targeted binding agents or antibodies comprising sequences whichhave at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acidsequence identity with any of the CDR1, CDR2 or CDR3s as shown in Table2 or Table 13, the contiguous sequences spanning the framework regionsand CDRs (specifically from FR1 through FR4 or CDR1 through CDR3) asshown in Table 2 or Table 13, the light or heavy chain sequencesdisclosed herein, or with the monoclonal antibodies disclosed herein.The percent identity of two amino acid sequences can be determined byany method known to one skilled in the art, including, but not limitedto, pairwise protein alignment. In one embodiment variants comprisechanges in the CDR sequences or light or heavy chain polypeptidesdisclosed herein that are naturally occurring or are introduced by invitro engineering of native sequences using recombinant DNA techniquesor mutagenesis techniques. Naturally occurring variants include thosewhich are generated in vivo in the corresponding germline nucleotidesequences during the generation of an antibody to a foreign antigen. Inone embodiment the derivative may be a heteroantibody, that is anantibody in which two or more antibodies are linked together.Derivatives include antibodies which have been chemically modified.Examples include covalent attachment of one or more polymers, such aswater-soluble polymers, N-linked, or O-linked carbohydrates, sugars,phosphates, and/or other such molecules. The derivatives are modified ina manner that is different from the naturally occurring or startingantibody, either in the type or location of the molecules attached.Derivatives further include deletion of one or more chemical groupswhich are naturally present on the antibody.

In one embodiment, the targeted binding agent is a bispecific antibody.A bispecific antibody is an antibody that has binding specificity for atleast two different epitopes. Methods for making bispecific antibodiesare known in the art. (See, for example, Millstein et al., Nature,305:537-539 (1983); Traunecker et al., EMBO J., 10:3655-3659 (1991);Suresh et al., Methods in Enzymology, 121:210 (1986); Kostelny et al.,J. Immunol., 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl Acad.Sci. USA, 90:6444-6448 (1993); Gruber et al., J. Immunol., 152:5368(1994); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;5,601,81; 95,731,168; 4,676,980; and 4,676,980, WO 94/04690; WO91/00360; WO 92/200373; WO 93/17715; WO 92/08802; and EP 03089.)

In some embodiments of the invention, the targeted binding agent orantibody has its sequence optimized by either mutating its non-germlineresidues to germline residues, and/or removing structural liabilities.In one embodiment, the invention includes a sequence comprising SEQ IDNO.: 6. In certain embodiments, SEQ ID NO.: 6 comprises any one of thecombinations of germline and non-germline residues indicated by each rowof Table 7. In some embodiments, SEQ ID NO: 6 comprises any one, anytwo, any three, any four, any five, any six or all six of the germlineresidues as indicated in Table 7. In certain embodiments, SEQ ID NO.: 6comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 7.

In other embodiments, the targeted binding agent or antibody is derivedfrom a germline sequence with VH3-33, 6-13 and JH4 domains, wherein oneor more residues has been mutated to yield the corresponding germlineresidue at that position. In certain embodiments, SEQ ID NO.: 24 cancomprise further modifications that include removing structuralliabilities. For example, in addition to germlining, the C33 can bemutated to a S. Thus, SEQ ID NO.: 24 can comprise any one of the uniquecombinations of germline and non-germline residues indicated by each rowof Table 10 and further include the mutation of C33 to a S.

A further embodiment of the invention is a targeted binding agent orantibody which competes for binding to DLL4. In another embodiment, theinvention is directed to a targeted binding agent or antibody whichcompetes with native DLL4 for binding to the Notch 1 or Notch 4receptor. In another embodiment the targeted binding agent or antibodycompetes for binding to Notch 1 with any one of fully human monoclonalantibodies 4B4 or 21H3, 2H10, 9G8, or 21H3RK. “Competes” indicates thatthe targeted binding agent or antibody competes for binding to Notch 1with any one of fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1,17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK, i.e. competitionis unidirectional.

Embodiments of the invention include a targeted binding agent orantibody which cross competes with any one of fully human monoclonalantibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3,1D4, or 21H3RK for binding to DLL4. “Cross competes” indicates that thetargeted binding agent or antibody competes for binding to Notch 1 withany one of fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1,17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK, and vice versa,i.e. competition is bidirectional.

A further embodiment of the invention is a targeted binding agent orantibody that binds to the same epitope on DLL4 as the targeted bindingagent or antibodies of the invention. Embodiments of the invention alsoinclude a targeted binding agent or antibody that binds to the sameepitope on DLL4 as any one of fully human monoclonal antibodies 4B4,2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.It is clear from the cross-competition analysis that the antibodies ofthe invention have different or partially overlapping epitopes. Forexample, 4B4 cross competes with 21H3RK and 21H3. Also 4B4 and 21H3 donot bind DLL4 under reducing and denaturating conditions while 9G8 and2H10 do suggestive of binding to different epitopes.

In certain embodiments, the epitope is comprised of at least oneextracellular, portion of the DLL4. The at least one specified epitope(for example, for 21H3 or 21H3RK or 4B4) can comprise any combination ofat least one amino acid sequence of at least 3 amino acid residues tothe entire specified portion of contiguous amino acids occurring in DLL4between amino acids 147-224, i.e.,ICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLC (SEQ ID NO:90). In one embodiment, the epitopeis at least 4 amino acid residues, at least 5 amino acid residues, atleast 6 amino acid residues, at least 7 amino acid residues, at least 8amino acid residues or at least 9 amino acid residues, at least 10 aminoacid residues, at least 15 amino acid residues, at least 20 amino acidresidues, at least 25 amino acid residues, at least 30 amino acidresidues, at least 40 amino acid residues or at least 50 amino acidresidues, at least 60 amino acid residues, at least 70 amino acidresidues, at least 75 amino acid residues, at least 76 amino acidresidues, or 77 amino acid residues of SEQ ID NO:90. In anotherembodiment, the epitope occurs in amino acid 187-201 of human DLL4,TGEYCQQPICLSGCH (SEQ ID NO:91). In one embodiment, the epitope is atleast 4 amino acid residues, at least 5 amino acid residues, at least 6amino acid residues, at least 7 amino acid residues, at least 8 aminoacid residues or at least 9 amino acid residues, at least 10 amino acidresidues, at least 11 amino acid residues, at least 12 amino acidresidues, at least 13 amino acid residues, at least 14 amino acidresidues, or 15 amino acid residues of SEQ ID NO:91.

In one embodiment, the invention includes mouse cross-reactiveantibodies of the antibodies disclosed herein. In one embodiment, thevariable regions of the antibodies are altered such that the antibodiescan bind mouse DLL4. Typically the mouse cross-reactive antibodies havesimilar properties to the antibodies disclosed herein, e.g., can bindDLL4 and can inhibit binding of DLL4 to a Notch receptor. In oneexample, the variable region of the 21H3RK antibody is altered such thatit can bind mouse DLL4, e.g., the heavy chain has the followingalternations: H31 Asn to Lys, H52a Ala to Pro, H97 Val to Thr, H100b Valto Trp and H100e Glu to Ala (see SEQ ID NO:84) and the light chain hasthe following alterations: L30 Ser to Asn and L93 Asp to Ser (see SEQ IDNO:85). In another embodiment, the heavy chain has the followingalternations: H30Thr to Ile, H31Asn to Met, H52a Ala to Pro, H100b Valto Trp and H100e Glu to Ala (see SEQ ID NO:86) and the light chain hasthe following alterations: L93 Asp to Ser (see SEQ ID NO:87). In yetanother embodiment, the heavy chain has the following alternations: H30Thr to Ile, H31 Asn to His, H100b Val to Trp and H100e Glu to Ala (seeSEQ ID NO:88). and the light chain has the following alterations: L30Ser to Asn and L93 Asp to Ser. (see SEQ ID NO:89).

Other embodiments of the invention include isolated nucleic acidmolecules encoding any of the targeted binding agents or antibodiesdescribed herein, vectors having isolated nucleic acid moleculesencoding the targeted binding agents or antibodies described herein or ahost cell transformed with any of such nucleic acid molecules.Embodiments of the invention include a nucleic acid molecule encoding afully human isolated targeted binding agent that specifically bind toDLL4 and inhibit binding of DLL4 to a Notch receptor. The invention alsoencompasses polynucleotides that hybridize under stringent or lowerstringency hybridization conditions, as defined herein, topolynucleotides that encode any of the targeted binding agents orantibodies described herein. Embodiments of the invention also include avector comprising the nucleic acid molecule encoding the binding agent.Additional embodiments include a host cell comprising the vector ofcomprising the nucleic acid molecule.

As known in the art, antibodies can advantageously be, for example,polyclonal, oligoclonal, monoclonal, chimeric, humanised, and/or fullyhuman antibodies.

It will be appreciated that embodiments of the invention are not limitedto any particular form of an antibody or method of generation orproduction. In some embodiments of the invention, the targeted bindingagent is a binding fragment of a fully human monoclonal antibody. Forexample, the targeted binding agent can be a full-length antibody (e.g.,having an intact human Fc region) or an antibody binding fragment (e.g.,a Fab, Fab′ or F(ab′)₂, FV or dAb). In addition, the antibodies can besingle-domain antibodies such as camelid or human single VH or VLdomains that bind to DLL4, such as a dAb fragment.

Embodiments of the invention described herein also provide cells forproducing these antibodies. Examples of cells include hybridomas, orrecombinantly created cells, such as Chinese hamster ovary (CHO) cells,variants of CHO cells (for example DG44) and NS0 cells that produceantibodies against DLL4. Additional information about variants of CHOcells can be found in Andersen and Reilly (2004) Current Opinion inBiotechnology 15, 456-462 which is incorporated herein in its entiretyby reference. The antibody can be manufactured from a hybridoma thatsecretes the antibody, or from a recombinantly engineered cell that hasbeen transformed or transfected with a gene or genes encoding theantibody.

In addition, one embodiment of the invention is a method of producing anantibody of the invention by culturing host cells under conditionswherein a nucleic acid molecule is expressed to produce the antibodyfollowed by recovering the antibody. It should be realised thatembodiments of the invention also include any nucleic acid moleculewhich encodes an antibody or fragment of an antibody of the inventionincluding nucleic acid sequences optimised for increasing yields ofantibodies or fragments thereof when transfected into host cells forantibody production.

A further embodiment herein includes a method of producing antibodiesthat specifically bind to DLL4 and inhibit the biological activity ofDLL4, by immunizing a mammal with cells expressing human DLL4, isolatedcell membranes containing human DLL4, purified human DLL4, or a fragmentthereof, and/or one or more orthologous sequences or fragments thereof.

In other embodiments the invention provides compositions, including atargeted binding agent or antibody of the invention or binding fragmentthereof, and a pharmaceutically acceptable carrier or diluent.

Still further embodiments of the invention include methods ofeffectively treating an animal suffering from a proliferative,angiogenic disease by administering to the animal a therapeuticallyeffective dose of a targeted binding agent that specifically binds toDLL4. In certain embodiments the method further comprises selecting ananimal in need of treatment for a tumor, cancer, and/or a cellproliferative disorder, and administering to the animal atherapeutically effective dose of a targeted binding agent thatspecifically binds to DLL4.

Still further embodiments of the invention include methods ofeffectively treating an animal suffering from a neoplastic disease byadministering to the animal a therapeutically effective dose of atargeted binding agent that specifically binds to DLL4. In certainembodiments the method further comprises selecting an animal in need oftreatment for a neoplastic disease, and administering to the animal atherapeutically effective dose of a targeted binding agent thatspecifically binds to DLL4.

Still further embodiments of the invention include methods ofeffectively treating an animal suffering from a malignant tumor byadministering to the animal a therapeutically effective dose of atargeted binding agent that specifically binds to DLL4. In certainembodiments the method further comprises selecting an animal in need oftreatment for a malignant tumor, and administering to the animal atherapeutically effective dose of a targeted binding agent thatspecifically binds to DLL4.

Still further embodiments of the invention include methods ofeffectively treating an animal suffering from a disease or conditionassociated with DLL4 expression by administering to the animal atherapeutically effective dose of a targeted binding agent thatspecifically binds to DLL4. In certain embodiments the method furthercomprises selecting an animal in need of treatment for a disease orcondition associated with DLL4 expression, and administering to theanimal a therapeutically effective dose of a targeted binding agent thatspecifically binds to DLL4.

A malignant tumor may be selected from the group consisting of:melanoma, small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, thyroid tumor, gastric (stomach)cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer,lung cancer, glioblastoma, endometrial cancer, kidney cancer, coloncancer, pancreatic cancer, esophageal carcinoma, head and neck cancers,mesothelioma, sarcomas, biliary (cholangiocarcinoma), small boweladenocarcinoma, pediatric malignancies and epidermoid carcinoma.

Treatable proliferative or angiogenic diseases include neoplasticdiseases, such as, melanoma, small cell lung cancer, non-small cell lungcancer, glioma, hepatocellular (liver) carcinoma, thyroid tumor, gastric(stomach) cancer, gallbladder cancer, prostate cancer, breast cancer,ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrialcancer, kidney cancer, colon cancer, pancreatic cancer, ovarian,esophageal carcinoma, head and neck cancers, mesothelioma, sarcomas,biliary (cholangiocarcinoma), small bowel adenocarcinoma, pediatricmalignancies, epidermoid carcinoma and leukaemia, including chronicmyelogenous leukaemia.

In one embodiment the present invention is suitable for use ininhibiting DLL4, in patients with a tumor, which is dependent alone, orin part, on DLL4.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from aproliferative, or angiogenic related disease. In certain embodiments theuse further comprises selecting an animal in need of treatment for aproliferative, or angiogenic-related disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a neoplasticdisease. In certain embodiments the use further comprises selecting ananimal in need of treatment for a neoplastic disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from anon-neoplastic disease. In certain embodiments the use further comprisesselecting an animal in need of treatment for a non-neoplastic disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a malignanttumor. In certain embodiments the use further comprises selecting ananimal in need of treatment for a malignant tumor.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a disease orcondition associated with DLL4 expression. In certain embodiments theuse further comprises selecting an animal in need of treatment for adisease or condition associated with DLL4 expression.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a proliferative orangiogenic-related disease.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a neoplastic disease.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a malignant tumor.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a disease or condition associatedwith DLL4 expression.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a DLL4 induced disease.

In one embodiment treatment of a

-   -   a proliferative or angiogenic-related disease;    -   a neoplastic disease;    -   a malignant tumor; or    -   a disease or condition associated with DLL4 expression; or    -   comprises managing, ameliorating, preventing, any of the        aforementioned diseases or conditions.

In one embodiment treatment of a neoplastic disease comprises inhibitionof tumor growth, tumor growth delay, regression of tumor, shrinkage oftumor, increased time to regrowth of tumor on cessation of treatment,increased time to tumor recurrence, slowing of disease progression.

In some embodiments of the invention, the animal to be treated is ahuman.

In some embodiments of the invention, the targeted binding agent is afully human monoclonal antibody.

In some embodiments of the invention, the targeted binding agent isselected from the group consisting of fully human monoclonal antibodies4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or21H3RK.

Embodiments of the invention include a conjugate comprising the targetedbinding agent as described herein, and a therapeutic agent. In someembodiments of the invention, the therapeutic agent is a toxin. In otherembodiments, the therapeutic agent is a radioisotope. In still otherembodiments, the therapeutic agent is a pharmaceutical composition.

In another aspect, a method of selectively killing a cancerous cell in apatient is provided. The method comprises administering a fully humanantibody conjugate to a patient. The fully human antibody conjugatecomprises an antibody that can bind to DLL4 and an agent. The agent iseither a toxin, a radioisotope, or another substance that will kill acancer cell. The antibody conjugate thereby selectively kills the cancercell.

In one aspect, a conjugated fully human antibody that specifically bindsto DLL4 is provided. Attached to the antibody is an agent, and thebinding of the antibody to a cell results in the delivery of the agentto the cell. In one embodiment, the above conjugated fully humanantibody binds to an extracellular domain of DLL4. In anotherembodiment, the antibody and conjugated toxin are internalised by a cellthat expresses DLL4. In another embodiment, the agent is a cytotoxicagent. In another embodiment, the agent is, for example saporin, orauristatin, pseudomonas exotoxin, gelonin, ricin, calicheamicin ormaytansine-based immunoconjugates, and the like. In still anotherembodiment, the agent is a radioisotope.

The targeted binding agent or antibody of the invention can beadministered alone, or can be administered in combination withadditional antibodies or chemotherapeutic drugs or radiation therapy.For example, a monoclonal, oligoclonal or polyclonal mixture of DLL4antibodies that block cell adhesion, invasion, angiogenesis orproliferation can be administered in combination with a drug shown toinhibit tumor cell proliferation.

Another embodiment of the invention includes a method of diagnosingdiseases or conditions in which an antibody as disclosed herein isutilised to detect the level of DLL4 in a patient or patient sample. Inone embodiment, the patient sample is blood or blood serum or urine. Infurther embodiments, methods for the identification of risk factors,diagnosis of disease, and staging of disease is presented which involvesthe identification of the expression and/or overexpression of DLL4 usinganti-DLL4 antibodies. In some embodiments, the methods compriseadministering to a patient a fully human antibody conjugate thatselectively binds to DLL4 on a cell. The antibody conjugate comprises anantibody that specifically binds to DLL4 and a label. The methodsfurther comprise observing the presence of the label in the patient. Arelatively high amount of the label will indicate a relatively high riskof the disease and a relatively low amount of the label will indicate arelatively low risk of the disease. In one embodiment, the label is agreen fluorescent protein.

The invention further provides methods for assaying the level of DLL4 ina patient sample, comprising contacting an antibody as disclosed hereinwith a biological sample from a patient, and detecting the level ofbinding between said antibody and DLL4 in said sample. In more specificembodiments, the biological sample is blood, plasma or serum.

Another embodiment of the invention includes a method for diagnosing acondition associated with the expression of DLL4 in a cell by contactingthe serum or a cell with an antibody as disclosed herein, and thereafterdetecting the presence of DLL4. In one embodiment the condition can be aproliferative, angiogenic, cell adhesion or invasion-related diseaseincluding, but not limited to, a neoplastic disease.

In another embodiment, the invention includes an assay kit for detectingDLL4 in mammalian tissues, cells, or body fluids to screen forDLL4-related diseases. The kit includes an antibody as disclosed hereinand a means for indicating the reaction of the antibody with DLL4, ifpresent. In one embodiment the antibody is a monoclonal antibody. In oneembodiment, the antibody that binds DLL4 is labelled. In anotherembodiment the antibody is an unlabelled primary antibody and the kitfurther includes a means for detecting the primary antibody. In oneembodiment, the means for detecting includes a labelled second antibodythat is an anti-immunoglobulin. The antibody may be labelled with amarker selected from the group consisting of a fluorochrome, an enzyme,a radionuclide and a radiopaque material.

In some embodiments, the targeted binding agents or antibodies asdisclosed herein can be modified to enhance their capability of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).In other embodiments, the targeted binding agents or antibodies can bemodified to enhance their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC). In yet otherembodiments, the targeted binding agents or antibodies as disclosedherein can be modified both to enhance their capability of activatingeffector cells and participating in antibody-dependent cytotoxicity(ADCC) and to enhance their capability of fixing complement andparticipating in complement-dependent cytotoxicity (CDC).

In some embodiments, the targeted binding agents or antibodies asdisclosed herein can be modified to reduce their capability of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).In other embodiments, the targeted binding agents or antibodies can bemodified to reduce their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC). In yet otherembodiments, the targeted binding agents or antibodies as disclosedherein can be modified both to reduce their capability of activatingeffector cells and participating in antibody-dependent cytotoxicity(ADCC) and to reduce their capability of fixing complement andparticipating in complement-dependent cytotoxicity (CDC).

In certain embodiments, the half-life of a targeted binding agent orantibody as disclosed herein and of compositions of the invention is atleast about 4 to 7 days. In certain embodiments, the mean half-life of atargeted binding agent or antibody as disclosed herein and ofcompositions of the invention is at least about 2 to 5 days, 3 to 6days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to 11 days,8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15to 19, or 16 to 20 days. In other embodiments, the mean half-life of atargeted binding agent or antibody as disclosed herein and ofcompositions of the invention is at least about 17 to 21 days, 18 to 22days, 19 to 23 days, 20 to 24 days, 21 to 25, days, 22 to 26 days, 23 to27 days, 24 to 28 days, 25 to 29 days, or 26 to 30 days. In stillfurther embodiments the half-life of a targeted binding agent orantibody as disclosed herein and of compositions of the invention can beup to about 50 days. In certain embodiments, the half-lives ofantibodies and of compositions of the invention can be prolonged bymethods known in the art. Such prolongation can in turn reduce theamount and/or frequency of dosing of the antibody compositions.Antibodies with improved in vivo half-lives and methods for preparingthem are disclosed in U.S. Pat. No. 6,277,375; and InternationalPublication Nos. WO 98/23289 and WO 97/3461.

In another embodiment, the invention provides an article of manufactureincluding a container. The container includes a composition containing atargeted binding agent or antibody as disclosed herein, and a packageinsert or label indicating that the composition can be used to treatcell adhesion, invasion, angiogenesis, and/or proliferation-relateddiseases, including, but not limited to, diseases characterised by theexpression or overexpression of DLL4.

In other embodiments, the invention provides a kit comprising acomposition containing a targeted binding agent or antibody as disclosedherein, and instructions to administer the composition to a subject inneed of treatment.

The present invention provides formulation of proteins comprising avariant Fc region. That is, a non-naturally occurring Fc region, forexample an Fc region comprising one or more non naturally occurringamino acid residues. Also encompassed by the variant Fc regions ofpresent invention are Fc regions which comprise amino acid deletions,additions and/or modifications.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. In oneembodiment, the Fc variant protein has enhanced serum half life relativeto comparable molecule.

In another embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid at one or more positions selected from the group consistingof 239, 330 and 332, as numbered by the EU index as set forth in Kabat.In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may further comprise additional non naturally occurring aminoacids at one or more positions selected from the group consisting of252, 254, and 256, as numbered by the EU index as set forth in Kabat. Ina specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat and at least one nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 252Y, 254T and 256E, as numbered by the EU indexas set forth in Kabat.

In another embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid at one or more positions selected from the group consistingof 234, 235 and 331, as numbered by the EU index as set forth in Kabat.In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 234F, 235F, 235Y, and331S, as numbered by the EU index as set forth in Kabat. In a furtherspecific embodiment, an Fc variant of the invention comprises the 234F,235F, and 331S non naturally occurring amino acid residues, as numberedby the EU index as set forth in Kabat. In another specific embodiment,an Fc variant of the invention comprises the 234F, 235Y, and 331S nonnaturally occurring amino acid residues, as numbered by the EU index asset forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 234F, 235F, 235Y, and 331S, as numbered by theEU index as set forth in Kabat; and at least one non naturally occurringamino acid at one or more positions are selected from the groupconsisting of 252Y, 254T and 256E, as numbered by the EU index as setforth in Kabat.

In another embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least a nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 239, 330 and 332, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 239D, 330L and 332E, as numbered by the EU indexas set forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant protein formulation, whereinthe Fc region comprises at least one non naturally occurring amino acidselected from the group consisting of 239D, 330L and 332E, as numberedby the EU index as set forth in Kabat and at least one non naturallyoccurring amino acid at one or more positions are selected from thegroup consisting of 252Y, 254T and 256E, as numbered by the EU index asset forth in Kabat.

In another embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least one nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 234, 235 and 331, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 234F, 235F, 235Y, and 331S, as numbered by theEU index as set forth in Kabat. Optionally, the Fc region may furthercomprise additional non naturally occurring amino acid at one or morepositions selected from the group consisting of 252, 254, and 256, asnumbered by the EU index as set forth in Kabat. In a specificembodiment, the present invention provides an Fc variant proteinformulation, wherein the Fc region comprises at least one non naturallyoccurring amino acid selected from the group consisting of 234F, 235F,235Y, and 331S, as numbered by the EU index as set forth in Kabat; andat least one non naturally occurring amino acid at one or more positionsare selected from the group consisting of 252Y, 254T and 256E, asnumbered by the EU index as set forth in Kabat.

Methods for generating non naturally occurring Fc regions are known inthe art. For example, amino acid substitutions and/or deletions can begenerated by mutagenesis methods, including, but not limited to,site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492(1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methodsand Applications”, Academic Press, San Diego, pp. 177-183 (1990)), andcassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably,site-directed mutagenesis is performed by the overlap-extension PCRmethod (Higuchi, in “PCR Technology: Principles and Applications for DNAAmplification”, Stockton Press, New York, pp. 61-70 (1989)). Thetechnique of overlap-extension PCR (Higuchi, ibid.) can also be used tointroduce any desired mutation(s) into a target sequence (the startingDNA). For example, the first round of PCR in the overlap-extensionmethod involves amplifying the target sequence with an outside primer(primer 1) and an internal mutagenesis primer (primer 3), and separatelywith a second outside primer (primer 4) and an internal primer (primer2), yielding two PCR segments (segments A and B). The internalmutagenesis primer (primer 3) is designed to contain mismatches to thetarget sequence specifying the desired mutation(s). In the second roundof PCR, the products of the first round of PCR (segments A and B) areamplified by PCR using the two outside primers (primers 1 and 4). Theresulting full-length PCR segment (segment C) is digested withrestriction enzymes and the resulting restriction fragment is clonedinto an appropriate vector. As the first step of mutagenesis, thestarting DNA (e.g., encoding an Fc fusion protein, an antibody or simplyan Fc region), is operably cloned into a mutagenesis vector. The primersare designed to reflect the desired amino acid substitution. Othermethods useful for the generation of variant Fc regions are known in theart (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO04/063351).

In some embodiments of the invention, the glycosylation patterns of theantibodies provided herein are modified to enhance ADCC and CDC effectorfunction. See Shields R L et al., (2002) JBC. 277:26733; Shinkawa T etal., (2003) JBC. 278:3466 and Okazaki A et al., (2004) J. Mol. Biol.,336: 1239. In some embodiments, an Fc variant protein comprises one ormore engineered glycoforms, i.e., a carbohydrate composition that iscovalently attached to the molecule comprising an Fc region. Engineeredglycoforms may be useful for a variety of purposes, including but notlimited to enhancing or reducing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example DIN-acetylglucosaminyltransferase III (GnTI11), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed. Methods for generating engineeredglycoforms are known in the art, and include but are not limited tothose described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davieset al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1;PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton,N.J.); GlycoMAb™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739;EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

It is also known in the art that the glycosylation of the Fc region canbe modified to increase or decrease effector function (see for examples,Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001,Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.);GlycoMAb™ glycosylation engineering technology (GLYCART biotechnologyAG, Zurich, Switzerland). Accordingly, in one embodiment the Fc regionsof the antibodies of the invention comprise altered glycosylation ofamino acid residues. In another embodiment, the altered glycosylation ofthe amino acid residues results in lowered effector function. In anotherembodiment, the altered glycosylation of the amino acid residues resultsin increased effector function. In a specific embodiment, the Fc regionhas reduced fucosylation. In another embodiment, the Fc region isafucosylated (see for examples, U.S. Patent Application Publication No.2005/0226867).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a bar graph showing the effects of IgG1 DLL4 antibodieson DLL4-stimulated inhibition of HUVEC proliferation. Data isrepresentative of n>2 independent experiments.

FIG. 2 depicts a bar graph showing the effects of IgG2/4 DLL4 antibodieson HUVEC tube formation in vitro as assessed by measurement of vessellength (mm) and # bifurcations.

FIG. 3 depicts a bar graph showing the effects of unlabeled anti-DLL4antibodies to displace the binding of Alexa-647 labeled 21H3RK at 0.1μg/ml as determined by FACS analysis

FIG. 4. depicts a graphic linear representation of twelve chimericDLL4/DLL1 variants. All variants encode the extracellular domains ofhuman DLL4 with human DLL1 replacing individual sub domains or combineddomain segments as depicted.

FIGS. 5A-5D depicts line graphs showing binding of 21H3RK to chimericknock out (“KO”) variants encoding DLL4 with segments of theextracellular domain substituted with the corresponding DLL1 domains.FIG. 5A depicts binding data of 21H3RK to chimeric knock out variantswhere the DLL4 N-ter1, N-ter2 or DSL segments of the extracellulardomain are substituted with the corresponding DLL1 domains. FIG. 5Bdepicts binding data of 21H3RK to chimeric knock out variants where theDLL4 EGF1, EGF12, EGF34 or EGF5678 segments of the extracellular domainare substituted with the corresponding DLL1 domains. FIG. 5C depictsbinding data of 21H3RK to chimeric knock out variants where the DLL4N-ter+DSL, DSL+EGF1, DSL+EGF12, or N-ter+DSL+EGF12 segments of theextracellular domain are substituted with the corresponding DLL1domains. FIG. 5D depicts binding data of 21H3RK to HuDLL4, HDLL1, oruntransfected cells.

FIGS. 6A-6C depicts line graphs showing binding of 21H3RK to chimericknock out (“KO”) variants encoding DLL4 with segments of theextracellular domain substituted with the corresponding DLL1 domains andchimeric knock in (“KI”) variants encoding DLL1 with regions substitutedwith DLL4 counterparts. FIG. 6A depicts binding data of 21H3RK tochimeric knock out variants where the DLL4 DSL+EGF1, A, B or C segmentsof the extracellular domain are substituted with the corresponding DLL1domains. FIG. 6B depicts binding data of 21H3RK to chimeric knock invariants where the DLL1 DSL, EGF1, DSL+EGF1 regions are substituted withthe corresponding DLL4 domains or to DLL4. FIG. 6C depicts binding dataof 21H3RK to DLL1 or untransfected cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the invention relate to a novel set of DLL4 blockingmolecules, such as, for example, antibodies, that inhibit notch receptorsignaling. Such molecules can be used as single agents, oralternatively, in combination with other binding antibodies/agents. Theycan also be used in combination with any standard or novel anti-canceragents.

Embodiments of the invention relate to targeted binding agents that bindto DLL4. In some embodiments, the targeted binding agents bind to DLL4and inhibit the binding of DLL4 to a Notch receptor (such as Notch 1,Notch 2, Notch 3, and/or Notch 4). In some embodiments, this binding canneutralize, block, inhibit, abrogate, or interfere with one or moreaspects of DLL4-associated effects. In one embodiment, the targetedbinding agents are monoclonal antibodies, or binding fragments thereof.Such monoclonal antibodies may be referred to as anti-DLL4 antibodiesherein.

Other embodiments of the invention include fully human anti-DLL4antibodies, and antibody preparations that are therapeutically useful.In one embodiment, preparations of the anti-DLL4 antibody of theinvention have desirable therapeutic properties, including strongbinding affinity for DLL4, ability to block DLL4 receptor-ligandinteractions, ability to block DLL4 mediated signaling, the ability topromote endothelial cell proliferation and formation of non-functionalblood vessels, ability to modulate pericyte recruitment to vessels,ability to inhibit tumor growth, ability to increase tumorhypoxia/necrosis, ability to alter endothelial tip/stalk cell fate, andability to modulate tumor cell survival or cancer stem cell survival andself renewal.

In addition, embodiments of the invention include methods of using theantibodies described herein for treating diseases. Anti-DLL4 antibodiesof the invention are useful for preventing DLL4-mediated tumorigenesisand tumor invasion of healthy tissue. In addition DLL4 antibodies can beuseful for treating diseases associated with angiogenesis such as oculardisease such as AMD, inflammatory disorders such as rheumatoidarthritis, and cardiovascular disease and sepsis as well as neoplasticdiseases. Any disease that is characterized by any type of malignanttumor, including metastatic cancers, lymphatic tumors, and bloodcancers, can also be treated by this inhibition mechanism. Exemplarycancers in humans include a bladder tumor, breast tumor, prostate tumor,basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer,brain and CNS cancer (e.g., glioma tumor), cervical cancer,choriocarcinoma, colon and rectum cancer, connective tissue cancer,cancer of the digestive system; endometrial cancer, esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer;intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; livercancer; lung cancer (e.g. small cell and non-small cell); lymphomaincluding Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma,neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, andpharynx); ovarian cancer; pancreatic cancer, retinoblastoma;rhabdomyosarcoma; rectal cancer, renal cancer, cancer of the respiratorysystem; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroidcancer; uterine cancer, cancer of the urinary system, as well as othercarcinomas and sarcomas. Malignant disorders commonly diagnosed in dogs,cats, and other pets include, but are not limited to, lymphosarcoma,osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma,adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor,bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma,neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma,Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma,osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma andrhabdomyosarcoma, genital squamous cell carcinoma, transmissiblevenereal tumor, testicular tumor, seminoma, Sertoli cell tumor,hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma),corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma,pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenalgland carcinoma, oral papillomatosis, hemangioendothelioma andcystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcomaand pulmonary squamous cell carcinoma. In rodents, such as a ferret,exemplary cancers include insulinoma, lymphoma, sarcoma, neuroma,pancreatic islet cell tumor, gastric MALT lymphoma and gastricadenocarcinoma. Neoplasias affecting agricultural livestock includeleukemia, hemangiopericytoma and bovine ocular neoplasia (in cattle);preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputialcarcinoma, connective tissue neoplasia and mastocytoma (in horses);hepatocellular carcinoma (in swine); lymphoma and pulmonary adenomatosis(in sheep); pulmonary sarcoma, lymphoma, Rous sarcoma,reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphomaand lymphoid leukosis (in avian species); retinoblastoma, hepaticneoplasia, lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemiaand swimbladder sarcoma (in fish), caseous lumphadenitis (CLA): chronic,infectious, contagious disease of sheep and goats caused by thebacterium Corynebacterium pseudotuberculosis, and contagious lung tumorof sheep caused by jaagsiekte.

Other embodiments of the invention include diagnostic assays forspecifically determining the quantity of DLL4 in a biological sample.The assay kit can include a targeted binding agent or antibody asdisclosed herein along with the necessary labels for detecting suchantibodies. These diagnostic assays are useful to screen for celladhesion, invasion, angiogenesis or proliferation-related diseasesincluding, but not limited to, neoplastic diseases.

Another aspect of the invention is an antagonist of the biologicalactivity of DLL4 wherein the antagonist binds to DLL4. In oneembodiment, the antagonist is a targeted binding agent, such as anantibody. The antagonist may be selected from an antibody describedherein, for example, antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8,21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

In one embodiment the antagonist of the biological activity of DLL4 maybind to DLL4 and thereby inhibit or suppress DLL4 binding to Notch,thereby inhibiting cell adhesion and/or invasion and/or angiogenesisand/or proliferation.

One embodiment is a targeted binding agent which binds to the sameepitope or epitopes as fully human monoclonal antibody 4B4, 2H10, 21F7,12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

One embodiment is an antibody which binds to the same epitope orepitopes as fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3,9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

One embodiment is a hybridoma that produces the targeted binding agentas described hereinabove. In one embodiment is a hybridoma that producesthe light chain and/or the heavy chain of the antibodies as describedhereinabove. In one embodiment the hybridoma produces the light chainand/or the heavy chain of a fully human monoclonal antibody. In anotherembodiment the hybridoma produces the light chain and/or the heavy chainof fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8,20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. Alternatively the hybridomamay produce an antibody which binds to the same epitope or epitopes asfully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8,21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

Another embodiment is a nucleic acid molecule encoding the targetedbinding agent as described hereinabove. In one embodiment is a nucleicacid molecule encoding the light chain or the heavy chain of an antibodyas described hereinabove. In one embodiment the nucleic acid moleculeencodes the light chain or the heavy chain of a fully human monoclonalantibody. Still another embodiment is a nucleic acid molecule encodingthe light chain or the heavy chain of a fully human monoclonal antibodyselected from antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3,1E4, 3A7, 4B3, 1D4, or 21H3RK.

Another embodiment of the invention is a vector comprising a nucleicacid molecule or molecules as described hereinabove, wherein the vectorencodes a targeted binding agent as defined hereinabove. In oneembodiment of the invention is a vector comprising a nucleic acidmolecule or molecules as described hereinabove, wherein the vectorencodes a light chain and/or a heavy chain of an antibody as definedhereinabove.

Yet another embodiment of the invention is a host cell comprising avector as described hereinabove. Alternatively the host cell maycomprise more than one vector.

In addition, one embodiment of the invention is a method of producing atargeted binding agent of the invention by culturing host cells underconditions wherein a nucleic acid molecule is expressed to produce thetargeted binding agent, followed by recovery of the targeted bindingagent. In one embodiment of the invention is a method of producing anantibody of the invention by culturing host cells under conditionswherein a nucleic acid molecule is expressed to produce the antibody,followed by recovery of the antibody.

In one embodiment the invention includes a method of making a targetedbinding agent by transfecting at least one host cell with at least onenucleic acid molecule encoding the targeted binding agent as describedhereinabove, expressing the nucleic acid molecule in the host cell andisolating the targeted binding agent. In one embodiment the inventionincludes a method of making an antibody by transfecting at least onehost cell with at least one nucleic acid molecule encoding the antibodyas described hereinabove, expressing the nucleic acid molecule in thehost cell and isolating the antibody.

According to another aspect, the invention includes a method ofantagonizing the biological activity of DLL4 by administering anantagonist as described herein. The method may include selecting ananimal in need of treatment for disease-related cell adhesion and/orinvasion and/or angiogenesis and/or proliferation, and administering tothe animal a therapeutically effective dose of an antagonist of thebiological activity of DLL4.

Another aspect of the invention includes a method of antagonizing thebiological activity of DLL4 by administering a targeted binding agent asdescribed hereinabove. The method may include selecting an animal inneed of treatment for disease-related cell adhesion and/or invasionand/or angiogenesis and/or proliferation, and administering to theanimal a therapeutically effective dose of a targeted binding agentwhich antagonises the biological activity of DLL4.

Another aspect of the invention includes a method of antagonizing thebiological activity of DLL4 by administering an antibody as describedhereinabove. The method may include selecting an animal in need oftreatment for disease-related cell adhesion and/or invasion and/orangiogenesis and/or proliferation, and administering to the animal atherapeutically effective dose of an antibody which antagonises thebiological activity of DLL4.

According to another aspect there is provided a method of treatingdisease-related cell adhesion and/or invasion and/or angiogenesis and/orproliferation in an animal by administering a therapeutically effectiveamount of an antagonist of the biological activity of DLL4. The methodmay include selecting an animal in need of treatment for disease-relatedcell adhesion and/or invasion and/or angiogenesis and/or proliferation,and administering to the animal a therapeutically effective dose of anantagonist of the biological activity of DLL4.

According to another aspect there is provided a method of treatingdisease-related cell adhesion and/or invasion and/or angiogenesis and/orproliferation in an animal by administering a therapeutically effectiveamount of a targeted binding agent which antagonizes the biologicalactivity of DLL4. The method may include selecting an animal in need oftreatment for disease-related cell adhesion and/or invasion and/orangiogenesis and/or proliferation, and administering to the animal atherapeutically effective dose of a targeted binding agent whichantagonises the biological activity of DLL4. The targeted binding agentcan be administered alone, or can be administered in combination withadditional antibodies or chemotherapeutic drugs or radiation therapy.

According to another aspect there is provided a method of treatingdisease-related cell adhesion and/or invasion and/or angiogenesis and/orproliferation in an animal by administering a therapeutically effectiveamount of an antibody which antagonizes the biological activity of DLL4.The method may include selecting an animal in need of treatment fordisease-related cell adhesion and/or invasion and/or angiogenesis and/orproliferation, and administering to the animal a therapeuticallyeffective dose of an antibody which antagonises the biological activityof DLL4. The antibody can be administered alone, or can be administeredin combination with additional antibodies or chemotherapeutic drugs orradiation therapy.

According to another aspect there is provided a method of treatingcancer in an animal by administering a therapeutically effective amountof an antagonist of the biological activity of DLL4. The method mayinclude selecting an animal in need of treatment for cancer, andadministering to the animal a therapeutically effective dose of anantagonist which antagonises the biological activity of DLL4. Theantagonist can be administered alone, or can be administered incombination with additional antibodies or chemotherapeutic drugs orradiation therapy.

According to another aspect there is provided a method of treatingcancer in an animal by administering a therapeutically effective amountof a targeted binding agent which antagonizes the biological activity ofDLL4. The method may include selecting an animal in need of treatmentfor cancer, and administering to the animal a therapeutically effectivedose of a targeted binding agent which antagonises the biologicalactivity of DLL4. The targeted binding agent can be administered alone,or can be administered in combination with additional antibodies orchemotherapeutic drugs or radiation therapy.

According to another aspect there is provided a method of treatingcancer in an animal by administering a therapeutically effective amountof an antibody which antagonizes the biological activity of DLL4. Themethod may include selecting an animal in need of treatment for cancer,and administering to the animal a therapeutically effective dose of anantibody which antagonises the biological activity of DLL4. The antibodycan be administered alone, or can be administered in combination withadditional antibodies or chemotherapeutic drugs or radiation therapy.

According to another aspect there is provided a method of reducing orinhibiting tumor cell proliferation, adhesion, invasion and/orangiogenesis, in an animal by administering a therapeutically effectiveamount of an antibody which antagonizes the biological activity of DLL4.The method may include selecting an animal in need of a reduction orinhibition of proliferation, cell adhesion, invasion and/orangiogenesis, and administering to the animal a therapeuticallyeffective dose of an antibody which antagonises the biological activityof DLL4. The antibody can be administered alone, or can be administeredin combination with additional antibodies or chemotherapeutic drugs orradiation therapy.

According to another aspect there is provided a method of reducing tumorgrowth and/or metastasis, in an animal by administering atherapeutically effective amount of an antibody which antagonizes thebiological activity of DLL4. The method may include selecting an animalin need of a reduction of tumor growth and/or metastasis, andadministering to the animal a therapeutically effective dose of anantibody which antagonises the biological activity of DLL4. The antibodycan be administered alone, or can be administered in combination withadditional antibodies or chemotherapeutic drugs or radiation therapy.

According to another aspect of the invention there is provided the useof an antagonist of the biological activity of DLL4 for the manufactureof a medicament for the treatment of disease-related cell adhesionand/or invasion and/or angiogenesis and/or proliferation. In oneembodiment the antagonist of the biological activity of DLL4 is atargeted binding agent of the invention. In one embodiment theantagonist of the biological activity of DLL4 is an antibody of theinvention.

According to another aspect of the invention there is provided anantagonist of the biological activity of DLL4 for use as a medicamentfor the treatment of disease-related cell adhesion and/or invasionand/or angiogenesis and/or proliferation. In one embodiment theantagonist of the biological activity of DLL4 is a targeted bindingagent of the invention. In one embodiment the antagonist of thebiological activity of DLL4 is an antibody of the invention.

According to another aspect of the invention there is provided the useof a targeted binding agent or an antibody which antagonizes thebiological activity of DLL4 for the manufacture of a medicament for thetreatment of disease-related cell adhesion and/or invasion and/orangiogenesis and/or proliferation.

According to another aspect of the invention there is provided atargeted binding agent or an antibody which antagonizes the biologicalactivity of DLL4 for use as a medicament for the treatment ofdisease-related cell adhesion and/or invasion and/or angiogenesis and/orproliferation.

According to another aspect of the invention there is provided the useof a targeted binding agent or an antibody which antagonizes thebiological activity of DLL4 for the manufacture of a medicament for thetreatment of disease-related cell adhesion and/or invasion and/orangiogenesis and/or proliferation.

According to another aspect of the invention there is provided anantibody which antagonizes the biological activity of DLL4 for use as amedicament for the treatment of disease-related cell adhesion and/orinvasion and/or angiogenesis and/or proliferation.

According to another aspect of the invention there is provided the useof an antagonist of the biological activity of DLL4 for the manufactureof a medicament for the treatment of cancer in a mammal. In oneembodiment the antagonist of the biological activity of DLL4 is atargeted binding agent of the invention. In one embodiment theantagonist of the biological activity of DLL4 is an antibody of theinvention.

According to another aspect of the invention there is provided anantagonist of the biological activity of DLL4 for use as a medicamentfor the treatment of cancer in a mammal. In one embodiment theantagonist of the biological activity of DLL4 is a targeted bindingagent of the invention. In one embodiment the antagonist of thebiological activity of DLL4 is an antibody of the invention.

According to another aspect of the invention there is provided the useof a targeted binding agent which antagonizes the biological activity ofDLL4 for the manufacture of a medicament for the treatment of cancer ina mammal.

According to another aspect of the invention there is provided atargeted binding agent which antagonizes the biological activity of DLL4for use as a medicament for the treatment of cancer in a mammal.

According to another aspect of the invention there is provided the useof an antibody which antagonizes the biological activity of DLL4 for themanufacture of a medicament for the treatment of cancer in a mammal.

According to another aspect of the invention there is provided anantibody which antagonizes the biological activity of DLL4 for use as amedicament for the treatment of cancer in a mammal.

According to another aspect there is provided the use of a targetedbinding agent or an antibody which antagonizes the biological activityof DLL4 for the manufacture of a medicament for the reduction orinhibition proliferation, cell adhesion, invasion and/or angiogenesis inan animal.

According to another aspect there is provided a targeted binding agentor an antibody which antagonizes the biological activity of DLL4 for useas a medicament for the reduction or inhibition proliferation, celladhesion, invasion and/or angiogenesis in an animal.

According to another aspect there is provided the use of a targetedbinding agent or an antibody which antagonizes the biological activityof DLL4 for the manufacture of a medicament for reducing tumor growthand/or metastasis, in an animal.

According to another aspect there is provided a targeted binding agentor an antibody which antagonizes the biological activity of DLL4 for useas a medicament for reducing tumor growth and/or metastasis, in ananimal.

In one embodiment the present invention is particularly suitable for usein antagonizing DLL4, in patients with a tumor, which is dependentalone, or in part, on DLL4.

According to another aspect of the invention there is provided apharmaceutical composition comprising an antagonist of the biologicalactivity of DLL4, and a pharmaceutically acceptable carrier. In oneembodiment the antagonist comprises an antibody. According to anotheraspect of the invention there is provided a pharmaceutical compositioncomprising an antagonist of the biological activity of DLL4, and apharmaceutically acceptable carrier. In one embodiment the antagonistcomprises an antibody.

In some embodiments, following administration of the antibody thatspecifically binds to DLL4, a clearing agent is administered, to removeexcess circulating antibody from the blood.

Anti-DLL4 antibodies are useful in the detection of DLL4 in patientsamples and accordingly are useful as diagnostics for disease states asdescribed herein. In addition, based on their ability to significantlyinhibit DLL4-mediated signaling activity (as demonstrated in theExamples below), anti-DLL4 antibodies have therapeutic effects intreating symptoms and conditions resulting from DLL4 expression. Inspecific embodiments, the antibodies and methods herein relate to thetreatment of symptoms resulting from DLL4 induced cell adhesion,invasion, angiogenesis, proliferation and/or intracellular signaling.Further embodiments involve using the antibodies and methods describedherein to treat cell adhesion, invasion, angiogenesis and/orproliferation-related diseases including neoplastic diseases, such as,melanoma, small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, thyroid tumor, gastric (stomach)cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer,lung cancer, glioblastoma, endometrial cancer, kidney cancer, coloncancer, and pancreatic cancer. The antibodies may also be useful intreating cell adhesion and/or invasion in arthritis, atherosclerosis anddiseases involving angiogenesis.

In one specific embodiment, the anti-DLL4 antibodies or targeted bindingagents can have therapeutic effects in treating solid tumors whosedevelopment relies on a small population of stem cells with the capacityto proliferate and efficiently give rise both to additional tumor stemcells, e.g., acute myeloid leukemia (AML) and breast tumors.

Another embodiment of the invention includes an assay kit for detectingDLL4 in mammalian tissues, cells, or body fluids to screen for celladhesion-, invasion-, angiogenesis- or proliferation related diseases.The kit includes a targeted binding agent that binds to DLL4 and a meansfor indicating the reaction of the targeted binding agent with DLL4, ifpresent. In one embodiment, the targeted binding agent that binds DLL4is labeled. In another embodiment the targeted binding agent is anunlabeled and the kit further includes a means for detecting thetargeted binding agent. Preferably the targeted binding agent is labeledwith a marker selected from the group consisting of a fluorochrome, anenzyme, a radionuclide and a radio-opaque material.

Another embodiment of the invention includes an assay kit for detectingDLL4 in mammalian tissues, cells, or body fluids to screen for celladhesion-, invasion-, angiogenesis or proliferation-related diseases.The kit includes an antibody that binds to DLL4 and a means forindicating the reaction of the antibody with DLL4, if present. Theantibody may be a monoclonal antibody. In one embodiment, the antibodythat binds DLL4 is labeled. In another embodiment the antibody is anunlabeled primary antibody and the kit further includes a means fordetecting the primary antibody. In one embodiment, the means includes alabeled second antibody that is an anti-immunoglobulin. Preferably theantibody is labeled with a marker selected from the group consisting ofa fluorochrome, an enzyme, a radionuclide and a radio-opaque material.

Further embodiments, features, and the like regarding the antibodies asdisclosed herein are provided in additional detail below.

Sequence Listing

Embodiments of the invention include the specific antibodies listedbelow in Table 1. This table reports the identification number of eachanti-DLL4 antibody, along with the SEQ ID number of the variable domainof the corresponding heavy chain and light chain genes and polypeptides,respectively. Each antibody has been given an identification number.

TABLE 1 mAb SEQ ID ID No.: Sequence NO: 4B4 Nucleotide sequence encodingthe variable region of the heavy chain 1 Amino acid sequence encodingthe variable region of the heavy chain 2 Nucleotide sequence encodingthe variable region of the light chain 3 Amino acid sequence encodingthe variable region of the light chain 4 2H10 Nucleotide sequenceencoding the variable region of the heavy chain 5 Amino acid sequenceencoding the variable region of the heavy chain 6 Nucleotide sequenceencoding the variable region of the light chain 7 Amino acid sequenceencoding the variable region of the light chain 8 21F7 Nucleotidesequence encoding the variable region of the heavy chain 9 Amino acidsequence encoding the variable region of the heavy chain 10 Nucleotidesequence encoding the variable region of the light chain 11 Amino acidsequence encoding the variable region of the light chain 12 12A1Nucleotide sequence encoding the variable region of the heavy chain 13Amino acid sequence encoding the variable region of the heavy chain 14Nucleotide sequence encoding the variable region of the light chain 15Amino acid sequence encoding the variable region of the light chain 1617F3 Nucleotide sequence encoding the variable region of the heavy chain17 Amino acid sequence encoding the variable region of the heavy chain18 Nucleotide sequence encoding the variable region of the light chain19 Amino acid sequence encoding the variable region of the light chain20 9G8 Nucleotide sequence encoding the variable region of the heavychain 21 Amino acid sequence encoding the variable region of the heavychain 22 Nucleotide sequence encoding the variable region of the lightchain 23 Amino acid sequence encoding the variable region of the lightchain 24 20G8 Nucleotide sequence encoding the variable region of theheavy chain 25 Amino acid sequence encoding the variable region of theheavy chain 26 Nucleotide sequence encoding the variable region of thelight chain 27 Amino acid sequence encoding the variable region of thelight chain 28 21H3 Nucleotide sequence encoding the variable region ofthe heavy chain 29 Amino acid sequence encoding the variable region ofthe heavy chain 30 Nucleotide sequence encoding the variable region ofthe light chain 31 Amino acid sequence encoding the variable region ofthe light chain 32 1E4 Nucleotide sequence encoding the variable regionof the heavy chain 33 Amino acid sequence encoding the variable regionof the heavy chain 34 Nucleotide sequence encoding the variable regionof the light chain 35 Amino acid sequence encoding the variable regionof the light chain 36 3A7 Nucleotide sequence encoding the variableregion of the heavy chain 37 Amino acid sequence encoding the variableregion of the heavy chain 38 Nucleotide sequence encoding the variableregion of the light chain 39 Amino acid sequence encoding the variableregion of the light chain 40 4B3 Nucleotide sequence encoding thevariable region of the heavy chain 41 Amino acid sequence encoding thevariable region of the heavy chain 42 Nucleotide sequence encoding thevariable region of the light chain 43 Amino acid sequence encoding thevariable region of the light chain 44 1D4 Nucleotide sequence encodingthe variable region of the heavy chain 45 Amino acid sequence encodingthe variable region of the heavy chain 46 Nucleotide sequence encodingthe variable region of the light chain 47 Amino acid sequence encodingthe variable region of the light chain 48 21H3RK Nucleotide sequenceencoding the variable region of the heavy chain 29 Amino acid sequenceencoding the variable region of the heavy chain 30 Nucleotide sequenceencoding the variable region of the light chain 49 Amino acid sequenceencoding the variable region of the light chain 50

Table 2 is a table comparing the antibody heavy chain regions to theircognate germ line heavy chain region and kappa light chain regions totheir cognate germ line lightchain region.

TABLE 2 Seq ID No Chain V D J FR1 CDR1  2 4B4.1VH QVLLIQSGAEVKKPG NYGVIASVQVSCKASGYTFT 51 Germline VH1-18 7-27 JH4 QVQLVQSGAEVKKPG SYGISASVKVSCKASGYTFT  4 4B4.1VL SYELTQPPSVSVSPG SGDALPKKYAY QTARITC 52Germline VL-3p JL2 SYELTQPPSVSVSPG SGDALPKKYAY QTARITC  6 2H10VHQVQLVESGGGVVQPG RHGMH RSLRLSCAASGFTFS 53 Germline VH3-33 6-13 JH4QVQLVESGGGVVQPG SYGMH RSLRLSCAASGFTFS  8 2H10VL SYELTQPPSVSVSPGSGDKLGDKYVC QTVSITC 54 Germline VL 3r JL2 SYELTQPPSVSVSPG SGDKLGDKYACQTASITC 10 21F7VH QVQLVQSGAEVKKPG NYGIS ASVKVSCQASGYTFT 55 GermlineVH1-18 6-15 JH4 QVQLVQSGAEVKKPG SYGIS ASVKVSCKASGYTFT 12 21F7VLSYELTQPPSVSVSPG SGDKLGDKYVC QTASITC 56 Germline VL 3r JL2SYELTQPPSVSVSPG SGDKLGDKYAC QTASITC 14 12A1VH QVQLVQSGAEVKRPG SYGITQSVKVSCKASGYTFT 57 Germline VH1-18 1-20 JH4 QVQLVQSGAEVKKPG SYGISASVKVSCKASGYTFT 16 12A1VL SYELTQPPSVSVSPG SGDTLPKKYAY QTARITC 58Germline VL-3p JL2 SYELTQPPSVSVSPG SGDALPKKYAY QTARITC 18 17F3VHQVQLVQSGAEVKKPG NYGIS ASVKVSCKASGYTFT 59 Germline VH1-18 2-2  JH4QVQLVQSGAEVKKPG SYGIS ASVKVSCKASGYTFT 20 17F3VL SYELTQPPSVSVSPGSGDKLGDKYVC QTASITC 60 Germline VL 3r JL2 SYELTQPPSVSVSPG SGDKLGDKYACQTASITC 22 9G8VH QLQLQESGPGLVKPS SSSSY ETLSLSCTVSGGSIS 61 GermlineVH4-39 4-23 JH3 QLQLQESGPGLVKPS SSSYY ETLSLTCTVSGGSIS 24 9G8VLSSELTQSPSVSVSPG SGDKLGDVYVC QTARITC 62 Germline VL 3r JL2SYELTQPPSVSVSPG SGDKLGDKYAC QTASITC 26 20G8VH QVQLVQSGAEVKKPG SYGISASVKVSCKASGYTFT 63 Germline VH1-18 2-15 JH3 QVQLVQSGAEVKKPG SYGISASVKVSCKASGYTFT 28 20G8VL QSVLTQPPSASGTPG SGSSSNIGSYY QRVTISC VY 64Germline VL1g JL2 QSVLTQPPSASGTPG SGSSSNIGSNY QRVTISC VY 30 21H3VHQVQLVQSGAEVKKPG NYGIT ASVKVSCKASGYTFT 65 Germline VH1-18 2-15 JH3QVQLVQSGAEVKKPG SYGIS ASVKVSCKASGYTFT 32 21H3VL QSVLTQPPSASGTPGSGSSSNIGSYF QRVTISC VY 66 Germline VL1g JL2 QSVLTQPPSASGTPG SGSSSNIGSNYQRVTISC VY 34 1E4VH QVQLVESGGGVVQPG SYGMH RSLRLSCAASGFTFS 67 GermlineVH3-33 2-2  JH6 QVQLVESGGGVVQPG SYGMH RSLRLSCAASGFTFS 36 1E4VLEIVLTQSPGTLSLSP RASQRVSSSYL GERATLSC T 68 Germline VH A27 JK5EIVLTQSPGTLSLSP RASQSVSSSYL GERATLSC A 38 3A7VH EVQVVESGGGLVQPG NYWMIGSLRLSCEASGFTFS 69 Germline VH3-07 1-26 JH6 EVQLVESGGGLVQPG SYWMSGSLRLSCAASGFTFS 40 3A7VL DIQMTQSPSSLSASV RASLDIRNDLG GDRVTITC 70Germline VK A30 JK3 DIQMTQSPSSLSASV RASQGIRNDLG GDRVTITC 42 4B3VHQVQLVQSGAEVRKSG TYDIN QSVSVSCKASGYSFT 71 Germline VH1-08 3-22 JH4QVQLVQSGAEVKKPG SYDIN QSVKVSCKASGYTFT 44 4B3VL DIQMTQSPSSLSASVRASQGISNYLA GDRVTITC 72 Germline VK L1 JK4 DIQMTQSPSSLSASV RASQGISNYLAGDRVTITC 46 1D4VH QLQLQESGPGLVKPS SSSYY RTLSLTCTVSGGSIS 73 GermlineVH4-39 6-19 JH3 QLQLQESGPGLVKPS SSSYY ETLSLTCTVSGGSIS 48 1D4VLSYELTQPPSVSVSPG SGDKLGDKFAC QTASITC 74 Germline VL 3r JL2SYELTQPPSVSVSPG SGDKLGDKYAC QTASITC Seq ID No FR2 CDR2 FR3 CDR3 FR4  2WVRQAPGQ WISAYNGNTN RVTVTSDTSTTTAYMEL ELGSSFDY WGQGTLVTVSS GLEYMGYAQKLQD RSLRSDDTAVYYCAR 51 WVRQAPGQ WISAYNGNTN RVTMTTDTSTSTAYMEL-LG-YFDY WGQGTLVTVSS GLEWMG YAQKLQG RSLRSDDTAVYYCAR  4 WYQQKSGQ EDIKRPSGIPERFSGSSSGTMATL FSTDNSGDHSV FGGGTKLTVL APVLVIY TISGAQVEDEADYYC 52WYQQKSGQ EDSKRPS GIPERFSGSSSGTMATL YSTDSSGNHVV FGGGTKLTVL APVLVIYTISGAQVEDEADYYC  6 WVRQAPGK VVWFDGSNIY RFTISRDNSKNTLYLQM DSRIAAADYWGQGTLVTVSS GLEWVA YADSVKG NSLRAEDTAMYYCAR 53 WVRQAPGK VIWYDGSNKYRFTISRDNSKNTLYLQM ---IAAADY WGQGTLVTVSS GLEWVA YADSVKG NSLRAEDTAVYYCAR 8 WYQQKPGQ QESKRPS GIPERFSGSSSGNTATL QTWDSSL-VV FGGGTKLTVL SPVLVIYTISGTQAMDEADYYC 54 WYQQKPGQ QDSKRPS GIPERFSGSNSGNTATL QAWDSSTAVVFGGGTKLTVL SPVLVIY TISGTQAMDEADYYC 10 WVRQPAGQ WINAYNGNTNRVTMTTDTSTNTAYMEL VAAAAFFDY WDQGTLVTVSS GLEWMG YAQNLQG RSLRSDDTAVYYCAR55 WVRQAPGQ WISAYNGNTN RVTMTTDTSTSTAYMEL --AAAYFDY WGQGTLVTVSS GLEWMGYAQKLQG RSLRSDDTAVYYCAR 12 WYQQKPGQ QDSKRPS GIPERFSGSNSGNTATL QAWDSST--VFGGGTKLTVL SPVLVIY TISGTQAMDEADYYC 56 WYQQKPGQ QDSKRPS GIPERFSGSNSGNTATLQAWDSSTAVV FGGGTKLTVL SPVLVIY TISGTQAMDEADYYC 14 WVRQAPGQ WISTYNGNTDRVTMTADISTSTAYMEL ERGSYFDY WGQGTLVTVSS GLEYMG YAQKFQG RSLRSDDTAVYYCAR 57WVRQAPGQ WISAYNGNTN RVTMTTDTSTSTAYMEL ER--YFDY WGQGTLVTVSS GLEWMGYAQKLQG RSLRSDDTAVYYCAR 16 WYQQKSGQ EDIKRPS GIPERFSGSSSGTMATLFSTDSGGNHK- FGGGTKLTVL APVLVIY TISGAQVEDEADYYC 58 WYQQKSGQ EDSKRPSGIPERFSGSSSGTMATL YSTDSSGNHVV FGGGTKLTVL APVLVIY TISGAQVEDEADYYC 18WVRQAPGQ WINAYNGNTN RVTMTTDTSTNTAYMEL VAPAAFFDY WDQGTLVTVSS GLEWMGYAQNLQG RSLRSDDTAVYYCAR 59 WVRQAPGQ WISAYNGNTN RVTMTTDTSTSTAYMELVVPAAYDFY WGQGTLVTVSS GLEWMG YAQKLQG RSLRSDDTAVYYCAR 20 WYQQKPGQ QDKNRPSGIPERFSGSNSGNTATL QAWDSST--V FGGGTKLTVL SPVLVIY IISGTQAMDEADYYC 60WYQQKPGQ QDSKRPS GIPERFSGSNSGNTATL QAWDSSTAVV FGGGTKLTVL SPVLVIYTISGTQAMDEADYYC 22 WGLIRQPP SIYYSGSTYY RVSISVDTSKNQFSLKL QGYGGHPDVFWGQGTMVTVSS GKGLEWIG SPSLKS SSVTAADTAVYFCAR DI 61 WGQIRQPP SIYYSGSTYYRVTISVDTSKNQFSLKL --YGG---AFDI WGQGTMVTVSS GKGLEWIG NPSLKSSSVTAADTAVYYCAR 24 WYQQKTGQ EDTKRPS GIPERFSGSNSGNTATL QAWDSTTAVIFGGGTKLTVL SPVLVIY TISGTQVMDEADYYC 62 WYQQKPGQ QDSKRPS GIPERFSGSNSGNTATLQAWDSSTAVV FGGGTKLTVL WPVLVIY TISGTQAMDEADYYC 26 WVRQAPGQ QISVYNGNTNRVTMTTDTSTSTAYMEV DRVPRIPRTTEAFDI WGRGTMVTVCS GLEWMG YAQKLQGRSLRSDDTAVYYCAR 63 WVRQAPGQ WISAYNGNTN RVTMTTDTSTSTAYMEL DIVVVVAAT--AFDIWGQGTMVTVSS GLEWMG YAQKLQG RSLRSDDTAVYYCAR 28 WYQQLPGT RDNQRPSGVPDRFSGSKSGTSASL AAWDDSLSGHWV FGGGTKLTVL APKLLIY AIRGLRSDDEADYYC 64WYQQLPGT RNNQRPS GVPDRFSGSKSGTSASL AAWDDSLSGV-V FGGGTKLTVL APKLLIYAISGLRSEDEADYYC 30 WVRQAPGQ WISAYNGNTN RVTVTTDTSTSTAYMEL DRVPRIPVTTEAFDIWGQGTMVTVSS GPEWMG YAQKLQD RSLRSDDTAVYYCAR 65 WVRQAPGQ WISAYNGNTNRVTMTTDTSTSTAYMEL DIVVVVAAT--AFDI WGQGTMVTVSS GLEWMG YAQKLQGRSLRSDDTAVYYCAR 32 WYQQLPGT RNNQRPS GVPDRFSGSESGTSASL AAWDDSLSGHWVFGGGTRLTVL APKLLIY AISGLRSEDEADYYC 66 WYQQLPGT RNNQRPS GVPDRFSGSKSGTSASLAAWDDSLSGV-V FGGGTKLTVL APKLLIY AISGLRSEDEADYYC 34 WVRQAPGK VTWYDGSNKYRFTISRDNSKNTLYLQM ENCSSTSCYYTVTTD WGQGTTVTVSS GLEWVA YADSVKGNSLRAEDTAVYYCAR YYGMDV 67 WVRQAPGK VIWYDGSNKY RFTISRDNSKNTLYLQM--CSSTSCY------ WGQGTTVTVSS GLEWVA YADSVKG NSLRAEDTAVYYCAR YYGMDV 36WYQQKPGQ GASIRAT GIPDRFSGSGSGTDFTL QQCYTSPIT FGQGTRLDIK APRLLIYTITRLEPEDFAVYFC 68 WYQQKPGQ GASSRAT GIPDRFSGSGSGTDFTL QQYGSSPITFGQGTRLEIK APRLLIY TISRLEPEDFAVYYC 38 WGRQAPGK SIKEDGSEKYRFTISRDNAKSSLYLQM DWELRGHYYYHGMDV WGQGTTVTVSS GLEWVA YVDSVKGNSLRAEDTAVYYCVR 69 WVRQAPGK NIKQDGSEKY RFTISRDNAKNSLYLQM -WEL--YYYYYGMDVWGQGTTVTVSS GLEWVA YVDSVKG NSLRAEDTAVYYCAR 40 WFLQKPGK AASSLQSGVPSRFSGSGSGTEFTL LQHRNYPFT FGPGTKVDFK APKRLIY TINSLQPEDFATYYC 70WYQQKPGK AASSLQS GVPSRFSGSGSGTEFTL LQHNSYPFT FGPGTKVDIK APKRLIYTISSLQPEDFATYYC 42 WVRQATGQ WMNPNSGYTD RVTLTRNMSIDTAYMEL AYYYDSSAYYLFDYWGQGTLVTVSS GLEWMG YAQKFQG SSLRSEDTAVYYCAR 71 WVRQATGQ WMNPNSGNTGRVTMTRNTSISTAYMEL -YYYDSSGYYYFDY WGQGTLVTVSS GLEWMG YAQKFQGSSLRSEDTAVYYCAR 44 WFQQKPGK AASSLQS GVPSKFSGSGSGTDFTL QQYISYPLTFGGGTKVEIK APKSLIY TISSLQPEDFATYFC 72 WFQQKPGK AASSLQS GVPSRFSGSGSGTDFTLQQYNSYPLT FGGGTKVEIK APKSLIY TISSLQPEDFATYYC 46 WGWIRQPP SFYYSRSTYYRVTISVDTSKNQFSLKL GSIAVPDAFDI WGQGTMVTVSS GKGLEWIG NPSLKSSSVTAADTAVHYCAR 73 WGQIRQPP SIYYSGSTYY RVTISVDTSKNQFSLKL --IAV--AFDIWGQGTMVTVSS GKGLEWIG NPSLKS SSVTAADTAVYYCAR 48 WYQQKPGH QDNKRPSGIPERFSGSNSGNSATL QAWDSNTA-V FGGGTKLTVL SPVLVVY TISGTQAMDEADYYC 74WYQQKPGQ QDSKRPS GIPERFSGSNSGNTATL QAWDSSTAVV FGGGTKLTVL SPVLVIYTISGTQAMDEADYYC

DEFINITIONS

Unless otherwise defined, scientific and technical terms used hereinshall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well known and commonly used in the art.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual (3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001)), which is incorporated herein by reference. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

An antagonist or inhibitor may be a polypeptide, nucleic acid,carbohydrate, lipid, small molecular weight compound, anoligonucleotide, an oligopeptide, RNA interference (RNAi), antisense, arecombinant protein, an antibody, or fragments thereof or conjugates orfusion proteins thereof. For a review of RNAi see Milhavet O, Gary D S,Mattson M P. (Pharmacol Rev. 2003 December; 55(4):629-48. Review) andantisense (see Opalinska J B, Gewirtz A M. (Sci STKE. 2003 Oct. 28; 2003(206):pe47.)

A compound refers to any small molecular weight compound with amolecular weight of less than about 2000 Daltons.

The term “DLL4” refers to the molecule that is DLL4 protein, also knownas Delta-like protein 4 precursor, Drosophila Delta homolog 4, hdelta2,MGC126344, or UNQ1895/PRO4341. The terms “neutralizing” or “inhibits”when referring to a targeted binding agent, such as an antibody, relatesto the ability of an antibody to eliminate, reduce, or significantlyreduce, the activity of a target antigen. Accordingly, a “neutralizing”anti-DLL4 antibody of the invention is capable of eliminating orsignificantly reducing the activity of DLL4. A neutralizing DLL4antibody may, for example, act by blocking the binding of a native DLL4to its receptor Notch, such as, for example, Notch 1 or Notch 4. Byblocking this binding, DLL4 signal-mediated activity is significantly,or completely, eliminated. Ideally, a neutralizing antibody against DLL4antagonism promotes EC proliferation. A neutralizing DLL4 antibody may,for example increase angiogenesis by promoting formation ofnon-functional vessels.

An “antagonist of the biological activity of DLL4” is capable ofeliminating, reducing or significantly reducing the activity of DLL4. An“antagonist of the biological activity of DLL4” is capable ofeliminating, reducing or significantly reducing DLL4 signaling. An“antagonist of the biological activity of DLL4” may eliminate orsignificantly reduce angiogenesis and/or proliferation.

“Reducing DLL4 signaling” encompasses a reduction of DLL4 signaling byat least 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% in comparison withthe level of signaling in the absence of a targeted binding agent,antibody or antagonist of the invention.

An “optimized” sequence is an antibody sequence (variable heavy or lightchain of any of the antibodies described herein) that has been mutatedsuch that the non-germline sequence is mutated back at one or moreresidues to the germline sequence, and can further include the removalof structural liabilities from the sequence such as glycosylation sitesor unpaired cysteines.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules and the human kappa lightchain immunoglobulin molecules, as well as antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as the kappa or lambda lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof. Preferred polypeptides in accordance with the inventionmay also comprise solely the human heavy chain immunoglobulin moleculesor fragments thereof.

The terms “native” or “naturally-occurring” as used herein as applied toan object refers to the fact that an object can be found in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by man in thelaboratory or otherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described that are in a relationship permitting them tofunction in their intended manner. For example, a control sequence“operably linked” to a coding sequence is connected in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, orRNA-DNA hetero-duplexes. The term includes single and double strandedforms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring linkages. Oligonucleotides are apolynucleotide subset generally comprising a length of 200 bases orfewer. Preferably, oligonucleotides are 10 to 60 bases in length andmost preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases inlength. Oligonucleotides are usually single stranded, e.g. for probes;although oligonucleotides may be double stranded, e.g. for use in theconstruction of a gene mutant. Oligonucleotides can be either sense orantisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof selectively hybridise to nucleic acid strands underhybridisation and wash conditions that minimise appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyconditions can be used to achieve selective hybridisation conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, orantibody fragments and a nucleic acid sequence of interest will be atleast 80%, and more typically with preferably increasing homologies ofat least 85%, 90%, 95%, 99%, and 100%.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) (0.9 M NaCl/90 mM NaCitrate, pH 7.0) at about 45° C. followed byone or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., highlystringent conditions such as hybridization to filter-bound DNA in 6×SSCat about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS atabout 60° C., or any other stringent hybridization conditions known tothose skilled in the art (see, for example, Ausubel, F. M. et al., eds.1989 Current Protocols in Molecular Biology, vol. 1, Green PublishingAssociates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to6.3.6 and 2.10.3). Two amino acid sequences are “homologous” if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least about 30 amino acids in length)are homologous, as this term is used herein, if they have an alignmentscore of more than 5 (in standard deviation units) using the programALIGN with the mutation data matrix and a gap penalty of 6 or greater.See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp.101-110 (Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. It should be appreciated that there can be differing regions ofhomology within two orthologous sequences. For example, the functionalsites of mouse and human orthologues may have a higher degree ofhomology than non-functional regions.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence.

In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA”.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the comparison window(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. The terms “substantial identity” asused herein denotes a characteristic of a polynucleotide or amino acidsequence, wherein the polynucleotide or amino acid comprises a sequencethat has at least 85 percent sequence identity, preferably at least 90to 95 percent sequence identity, more preferably at least 99 percentsequence identity, as compared to a reference sequence over a comparisonwindow of at least 18 nucleotide (6 amino acid) positions, frequentlyover a window of at least 24-48 nucleotide (8-16 amino acid) positions,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the left-hand direction is the aminoterminal direction and the right-hand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions that are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99% sequence identity to theantibodies or immunoglobulin molecules described herein. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat have related side chains. Genetically encoded amino acids aregenerally divided into families: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) non-polar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: serine and threonineare an aliphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding function or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the antibodies described herein.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

In general, cysteine residues in proteins are either engaged incysteine-cysteine disulfide bonds or sterically protected from thedisulfide bond formation when they are a part of folded protein region.Disulfide bond formation in proteins is a complex process, which isdetermined by the redox potential of the environment and specializedthiol-disulfide exchanging enzymes (Creighton, Methods Enzymol. 107,305-329, 1984; Houee-Levin, Methods Enzymol. 353, 35-44, 2002). When acysteine residue does not have a pair in protein structure and is notsterically protected by folding, it can form a disulfide bond with afree cysteine from solution in a process known as disulfide shuffling.In another process known as disulfide scrambling, free cysteines mayalso interfere with naturally occurring disulfide bonds (such as thosepresent in antibody structures) and lead to low binding, low biologicalactivity and/or low stability.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991), which are each incorporatedherein by reference.

Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds.

The term “CDR region” or “CDR” is intended to indicate the hypervariableregions of the heavy and light chains of an antibody which conferantigen-binding specificity to the antibody. CDRs may be definedaccording to the Kabat system (Kabat, E. A. et al. (1991) Sequences ofProteins of Immunological Interest, 5th Edition. US Department of Healthand Human Services, Public Service, NIH, Washington), and latereditions. An antibody typically contains 3 heavy chain CDRs and 3 lightchain CDRs. The term CDR or CDRs is used here in order to indicate,according to the case, one of these regions or several, or even thewhole, of these regions which contain the majority of the amino acidresidues responsible for the binding by affinity of the antibody for theantigen or the epitope which it recognises.

The third CDR of the heavy chain (HCDR3) has a greater size variability(greater diversity essentially due to the mechanisms of arrangement ofthe genes which give rise to it). It may be as short as 2 amino acidsalthough the longest size known is 26. CDR length may also varyaccording to the length that can be accommodated by the particularunderlying framework. Functionally, HCDR3 plays a role in part in thedetermination of the specificity of the antibody (Segal et al., PNAS,71:4298-4302, 1974, Amit et al., Science, 233:747-753, 1986, Chothia etal., J. Mol. Biol., 196:901-917, 1987, Chothia et al., Nature,342:877-883, 1989, Caton et al., J. Immunol., 144:1965-1968, 1990,Sharon et al., PNAS, 87:4814-4817, 1990, Sharon et al., J. Immunol.,144:4863-4869, 1990, Kabat et al., J. Immunol., 147:1709-1719, 1991).

The term a “set of CDRs” referred to herein comprises CDR1, CDR2 andCDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a setof LCDRs refers to LCDR1, LCDR2 and LCDR3.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in targeting agents and antibodies for DLL4 can beobtained by means of methods of sequence alteration or mutation andscreening for antigen targeting with desired characteristics. Examplesof desired characteristics include but are not limited to: increasedbinding affinity for antigen relative to known antibodies which arespecific for the antigen; increased neutralisation of an antigenactivity relative to known antibodies which are specific for the antigenif the activity is known; specified competitive ability with a knownantibody or ligand to the antigen at a specific molar ratio; ability toimmunoprecipitate ligand-receptor complex; ability to bind to aspecified epitope; linear epitope, e.g. peptide sequence identifiedusing peptide-binding scan, e.g. using peptides screened in linearand/or constrained conformation; conformational epitope, formed bynon-continuous residues; ability to modulate a new biological activityof DLL4, or downstream molecule; ability to bind and/or neutralise DLL4and/or for any other desired property.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and antigen binding sitesare available in the art. Variants of antibody molecules disclosedherein may be produced and used in the present invention. Following thelead of computational chemistry in applying multivariate data analysistechniques to the structure/property-activity relationships (Wold, etal. Multivariate data analysis in chemistry. Chemometrics—Mathematicsand Statistics in Chemistry (Ed.: B. Kowalski), D. Reidel PublishingCompany, Dordrecht, Holland, 1984) quantitative activity-propertyrelationships of antibodies can be derived using well-known mathematicaltechniques, such as statistical regression, pattern recognition andclassification (Norman et al. Applied Regression Analysis.Wiley-Interscience; 3rd edition (April 1998); Kandel, Abraham & Backer,Eric. Computer-Assisted Reasoning in Cluster Analysis. Prentice HallPTR, (May 11, 1995); Krzanowski, Wojtek. Principles of MultivariateAnalysis: A User's Perspective (Oxford Statistical Science Series, No 22(Paper)). Oxford University Press; (December 2000); Witten, Ian H. &Frank, Eibe. Data Mining Practical Machine Learning Tools and Techniqueswith Java Implementations. Morgan Kaufmann; (Oct. 11, 1999); DenisonDavid G. T. (Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F.M. Smith. Bayesian Methods for Nonlinear Classification and Regression(Wiley Series in Probability and Statistics). John Wiley & Sons; (July2002); Ghose, Arup K. & Viswanadhan, Vellarkad N. Combinatorial LibraryDesign and Evaluation Principles, Software, Tools, and Applications inDrug Discovery). In some cases the properties of antibodies can bederived from empirical and theoretical models (for example, analysis oflikely contact residues or calculated physicochemical property) ofantibody sequence, functional and three-dimensional structures and theseproperties can be considered singly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis typically formed by six loops of polypeptide: three from the lightchain variable domain (VL) and three from the heavy chain variabledomain (VH). Analysis of antibodies of known atomic structure haselucidated relationships between the sequence and three-dimensionalstructure of antibody combining sites. These relationships imply that,except for the third region (loop) in VH domains, binding site loopshave one of a small number of main-chain conformations: canonicalstructures. The canonical structure formed in a particular loop has beenshown to be determined by its size and the presence of certain residuesat key sites in both the loop and in framework regions.

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimisation experiments. In astructural approach, a model can be created of the antibody moleculeusing any freely available or commercial package, such as WAM. A proteinvisualisation and analysis software package, such as Insight II(Accelrys, Inc.) or Deep View may then be used to evaluate possiblesubstitutions at each position in the CDR. This information may then beused to make substitutions likely to have a minimal or beneficial effecton activity or confer other desirable properties.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to DLL4, under suitablebinding conditions, (2) ability to block appropriate VEGF/DLL4 binding,or (3) ability to inhibit DLL4 receptor tyrosine kinase activity.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference).Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

An antibody may be oligoclonal, a polyclonal antibody, a monoclonalantibody, a chimeric antibody, a CDR-grafted antibody, a multi-specificantibody, a bi-specific antibody, a catalytic antibody, a chimericantibody, a humanized antibody, a fully human antibody, ananti-idiotypic antibody and antibodies that can be labeled in soluble orbound form as well as fragments, variants or derivatives thereof, eitheralone or in combination with other amino acid sequences provided byknown techniques. An antibody may be from any species.

As used herein, the terms “antibody” and “antibodies” (immunoglobulins)encompass monoclonal antibodies (including full-length monoclonalantibodies), polyclonal antibodies, camelised antibodies and chimericantibodies. As used herein, the term “antibody” or “antibodies” refersto a polypeptide or group of polypeptides that are comprised of at leastone binding domain that is formed from the folding of polypeptide chainshaving three-dimensional binding spaces with internal surface shapes andcharge distributions complementary to the features of an antigenicdeterminant of an antigen. chain. Native antibodies are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges. Each heavy chain hasat one end a variable domain (VH) followed by a number of constantdomains. Each light chain has a variable domain at one end (VL) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Light chains are classified as either lambda chains orkappa chains based on the amino acid sequence of the light chainconstant region. The variable domain of a kappa light chain may also bedenoted herein as VK. The term “variable region” may also be used todescribe the variable domain of a heavy chain or light chain. Particularamino acid residues are believed to form an interface between the lightand heavy chain variable domains. The variable regions of eachlight/heavy chain pair form an antibody binding site. Such antibodiesmay be derived from any mammal, including, but not limited to, humans,monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.

The term “antibody” or “antibodies” includes binding fragments of theantibodies of the invention, exemplary fragments include single-chainFvs (scFv), single-chain antibodies, single domain antibodies, domainantibodies, Fv fragments, Fab fragments, F(ab′) fragments, F(ab′)2fragments, antibody fragments that exhibit the desired biologicalactivity, disulfide-stabilised variable region (dsFv), dimeric variableregion (Diabody), anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id antibodies to antibodies of the invention), intrabodies, linearantibodies, single-chain antibody molecules and multispecific antibodiesformed from antibody fragments and epitope-binding fragments of any ofthe above. In particular, antibodies include immunoglobulin moleculesand immunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site. Immunoglobulin moleculescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Digestion of antibodies with the enzyme, papain, results in twoidentical antigen-binding fragments, known also as “Fab” fragments, anda “Fc” fragment, having no antigen-binding activity but having theability to crystallize. Digestion of antibodies with the enzyme, pepsin,results in the a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)₂ fragment has the ability to crosslink antigen.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites. This regionconsists of a dimer of one heavy and one light chain variable domain intight, non-covalent or covalent association. It is in this configurationthat the three CDRs of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six CDRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree CDRs specific for an antigen) has the ability to recognize andbind antigen, although at a lower affinity than the entire binding site.

“Fab” when used herein refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

“dAb” when used herein refers to a fragment of an antibody that is thesmallest functional binding unit of a human antibodies. A “dAb” is asingle domain antibody and comprises either the variable domain of anantibody heavy chain (VH domain) or the variable domain of an antibodylight chain (VL domain). Each dAb contains three of the six naturallyoccurring CDRs (Ward et al., Binding activities of a repertoire ofsingle immunoglobulin variable domains secreted from Escherichia coli.Nature 341, 544-546 (1989); Holt, et al., Domain antibodies: protein fortherapy, Trends Biotechnol. 21, 484-49 (2003)). With molecular weightsranging from 11 to 15 kDa, they are four times smaller than a fragmentantigen binding (Fab)₂ and half the size of a single chain Fv (scFv)molecule.

“Camelid” when used herein refers to antibody molecules are composed ofheavy-chain dimers which are devoid of light chains, but neverthelesshave an extensive antigen-binding repertoire (Hamers-Casterman C,Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa E B, BendahmanN, Hamers R (1993) Naturally occurring antibodies devoid of lightchains. Nature 363:446-448).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (Ward,E. S. et al., (1989) Nature 341, 544-546) the Fab fragment consisting ofVL, VH, CL and CH1 domains; (McCafferty et al (1990) Nature, 348,552-554) the Fd fragment consisting of the VH and CH1 domains; (Holt etal (2003) Trends in Biotechnology 21, 484-490) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty etal (1990) Nature, 348, 552-554, Holt et al (2003) Trends inBiotechnology 21, 484-490], which consists of a VH or a VL domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al, (1988) Science, 242, 423-426, Huston et al,(1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804;Holliger, P. (1993) et al, Proc. Natl. Acad. Sci. USA 90 6444-6448). Fv,scFv or diabody molecules may be stabilised by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter, Y. et al,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (Hu, S. et al, (1996) CancerRes., 56, 3055-3061). Other examples of binding fragments are Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in segments called Complementarity Determining Regions(CDRs) both in the light chain and the heavy chain variable domains. Themore highly conserved portions of the variable domains are called theframework regions (FR). The variable domains of native heavy and lightchains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see, Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are generally not involved directly in antigen binding, but mayinfluence antigen binding affinity and may exhibit various effectorfunctions, such as participation of the antibody in ADCC, CDC, and/orapoptosis.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are associated with its binding toantigen. The hypervariable regions encompass the amino acid residues ofthe “complementarity determining regions” or “CDRs” (e.g., residues24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domainand residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chainvariable domain; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, J. Mol. Biol., 196:901-917(1987)). “Framework” or “FR” residues are those variable domain residuesflanking the CDRs. FR residues are present in chimeric, humanized,human, domain antibodies, diabodies, vaccibodies, linear antibodies, andbispecific antibodies.

As used herein, targeted binding agent, targeted binding protein,specific binding protein and like terms refer to an antibody, or bindingfragment thereof that preferentially binds to a target site. In oneembodiment, the targeted binding agent is specific for only one targetsite. In other embodiments, the targeted binding agent is specific formore than one target site. In one embodiment, the targeted binding agentmay be a monoclonal antibody and the target site may be an epitope.

“Binding fragments” of an antibody are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, dAb and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounter-receptor when an excess of antibody reduces the quantity ofreceptor bound to counter-receptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and may, but not always, havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≦1 μM, preferably ≦100nM and most preferably ≦10 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

“Active” or “activity” in regard to an DLL4 polypeptide refers to aportion of an DLL4 polypeptide that has a biological or an immunologicalactivity of a native DLL4 polypeptide. “Biological” when used hereinrefers to a biological function that results from the activity of thenative DLL4 polypeptide. A preferred DLL4 biological activity includes,for example, DLL4 induced cell adhesion and invasion and/or angiogenesisand/or proliferation.

“Mammal” when used herein refers to any animal that is considered amammal. Preferably, the mammal is human.

“Animal” when used herein encompasses animals considered a mammal.Preferably the animal is human.

The term “mAb” refers to monoclonal antibody.

“Liposome” when used herein refers to a small vesicle that may be usefulfor delivery of drugs that may include the DLL4 polypeptide of theinvention or antibodies to such an DLL4 polypeptide to a mammal.

“Label” or “labeled” as used herein refers to the addition of adetectable moiety to a polypeptide, for example, a radiolabel,fluorescent label, enzymatic label chemiluminescent labeled or abiotinyl group. Radioisotopes or radionuclides may include ³H, ¹⁴C, ¹⁵N,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, fluorescent labels may includerhodamine, lanthanide phosphors or FITC and enzymatic labels may includehorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase.

Additional labels include, by way of illustration and not limitation:enzymes, such as glucose-6-phosphate dehydrogenase (“G6PDH”),alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonicanhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase andperoxidase; dyes; additional fluorescent labels or fluorescers include,such as fluorescein and its derivatives, fluorochrome, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;fluorophores such as lanthanide cryptates and chelates e.g. Europium etc(Perkin Elmer and Cis Biointernational); chemoluminescent labels orchemiluminescers, such as isoluminol, luminol and the dioxetanes;sensitisers; coenzymes; enzyme substrates; particles, such as latex orcarbon particles; metal sol; crystallite; liposomes; cells, etc., whichmay be further labelled with a dye, catalyst or other detectable group;molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; toxinmoieties, such as for example a toxin moiety selected from a group ofPseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof),Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinumtoxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g.ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxicfragment thereof, pokeweed antiviral toxin or a cytotoxic fragmentthereof and bryodin 1 or a cytotoxic fragment thereof.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (incorporatedherein by reference).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” includes human and veterinary subjects.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells thatexpress Ig Fc receptors (FcRs) (e.g. Natural Killer (NK) cells,monocytes, neutrophils, and macrophages) recognise bound antibody on atarget cell and subsequently cause lysis of the target cell. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγRI, FcγRII and FcγRIII FcRs expression onhematopoietic cells is summarised in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. No. 5,500,362, or 5,821,337 can be performed. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells. Alternatively, or additionally, ADCCactivity of the molecule of interest can be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1988). “Complement dependent cytotoxicity” and “CDC” referto the mechanism by which antibodies carry out their cell-killingfunction. It is initiated by the binding of C1q, a constituent of thefirst component of complement, to the Fc domain of Igs, IgG or IgM,which are in complex with antigen (Hughs-Jones, N. C., and B. Gardner.1979. Mol. Immunol. 16:697). C1q is a large, structurally complexglycoprotein of ˜410 kDa present in human serum at a concentration of 70μg/ml (Cooper, N. R. 1985. Adv. Immunol. 37:151). Together with twoserine proteases, C1r and C1s, C1q forms the complex C1, the firstcomponent of complement. At least two of the N-terminal globular headsof C1q must be bound to the Fc of Igs for C1 activation, hence forinitiation of the complement cascade (Cooper, N. R. 1985. Adv. Immunol.37:151).

The term “antibody half-life” as used herein means a pharmacokineticproperty of an antibody that is a measure of the mean survival time ofantibody molecules following their administration. Antibody half-lifecan be expressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body or a specificcompartment thereof, for example, as measured in serum or plasma, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

The term “isotype” refers to the classification of an antibody's heavyor light chain constant region. The constant domains of antibodies arenot involved in binding to antigen, but exhibit various effectorfunctions. Depending on the amino acid sequence of the heavy chainconstant region, a given human antibody or immunoglobulin can beassigned to one of five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM. Several of these classes may be further divided intosubclasses (isotypes), e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma3), and IgG4 (gamma 4), and IgA1 and IgA2. The heavy chain constantregions that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. Of the various human immunoglobulin classes, only humanIgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. HumanIgG1 and IgG3 are known to mediate in humans. Human light chain constantregions may be classified into two major classes, kappa and lambda.

If desired, the isotype of an antibody that specifically binds DLL4 canbe switched, for example to take advantage of a biological property of adifferent isotype. For example, in some circumstances it can bedesirable in connection with the generation of antibodies as therapeuticantibodies against DLL4 that the antibodies be capable of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).There are a number of isotypes of antibodies that are capable of thesame, including, without limitation, the following: murine IgM, murineIgG2a, murine IgG2b, murine IgG3, human IgM, human IgA, human IgG1, andhuman IgG3. In other embodiments it can be desirable in connection withthe generation of antibodies as therapeutic antibodies against DLL4 thatthe antibodies be capable of binding Fc receptors on effector cells andparticipating in antibody-dependent cytotoxicity (ADCC). There are anumber of isotypes of antibodies that are capable of the same,including, without limitation, the following: murine IgG2a, murineIgG2b, murine IgG3, human IgG1, and human IgG3. It will be appreciatedthat antibodies that are generated need not initially possess such anisotype but, rather, the antibody as generated can possess any isotypeand the antibody can be isotype switched thereafter using conventionaltechniques that are well known in the art. Such techniques include theuse of direct recombinant techniques (see e.g., U.S. Pat. No.4,816,397), cell-cell fusion techniques (see e.g., U.S. Pat. Nos.5,916,771 and 6,207,418), among others.

By way of example, the anti-DLL4 antibodies discussed herein are fullyhuman antibodies. If an antibody possessed desired binding to DLL4, itcould be readily isotype switched to generate a human IgM, human IgG1,or human IgG3 isotype, while still possessing the same variable region(which defines the antibody's specificity and some of its affinity).Such molecule would then be capable of fixing complement andparticipating in CDC and/or be capable of binding to Fc receptors oneffector cells and participating in ADCC.

“Whole blood assays” use unfractionated blood as a source of naturaleffectors. Blood contains complement in the plasma, together withFcR-expressing cellular effectors, such as polymorphonuclear cells(PMNs) and mononuclear cells (MNCs). Thus, whole blood assays allowsimultaneous evaluation of the synergy of both ADCC and CDC effectormechanisms in vitro.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Stated in anotherway, a “therapeutically effective” amount is an amount that providessome alleviation, mitigation, and/or decrease in at least one clinicalsymptom. Clinical symptoms associated with the disorders that can betreated by the methods of the invention are well-known to those skilledin the art. Further, those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject.

The term “and/or” as used herein is to be taken as specific disclosureof each of the two specified features or components with or without theother. For example “A and/or B” is to be taken as specific disclosure ofeach of (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site.

Thus, an intact antibody has two binding sites. Except in bifunctionalor bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called CDRs. The CDRs from the two chains of each pair are alignedby the framework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FRE CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992). Bispecific antibodies do notexist in the form of fragments having a single binding site (e.g., Fab,Fab′, and Fv).

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although a VH or VL domain alone may be used tobind antigen. The VH domain (see Table 2) may be paired with the VLdomain (see Table 2), so that an antibody antigen-binding site is formedcomprising both the VH and VL domains.

Human Antibodies and Humanization of Antibodies

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of functional human antibodyloci into a rodent, other mammal or animal so that the rodent, othermammal or animal produces fully human antibodies.

One method for generating fully human antibodies is through the use ofXenoMouse® strains of mice that have been engineered to contain up tobut less than 1000 kb-sized germline configured fragments of the humanheavy chain locus and kappa light chain locus. See Mendez et al. NatureGenetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.188:483-495 (1998). The XenoMouse® strains are available from Amgen,Inc. (Fremont, Calif., U.S.A).

Such mice, then, are capable of producing human immunoglobulin moleculesand antibodies and are deficient in the production of murineimmunoglobulin molecules and antibodies. Technologies utilised forachieving the same are disclosed in U.S. patent application Ser. No.08/759,620, filed Dec. 3, 1996 and International Patent Application Nos.WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21,2000, the disclosures of which are hereby incorporated by reference. Seealso Mendez et al. Nature Genetics 15:146-156 (1997), the disclosure ofwhich is hereby incorporated by reference.

The production of the XenoMouse® strains of mice is further discussedand delineated in U.S. patent application Ser. No. 07/466,008, filedJan. 12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No.07/919,297, filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30,1992, Ser. No. 08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848,filed Aug. 27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No.08/376,279, filed Jan. 20, 1995, Ser. No. 08/430, 938, filed Apr. 27,1995, Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582,filed Jun. 5, 1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No.08/462,837, filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995,Ser. No. 08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun.5, 1995, Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752,filed Oct. 2, 1996, Ser. No. 08/759,620, filed Dec. 3, 1996, U.S.Publication 2003/0093820, filed Nov. 30, 2001 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoEuropean Patent No., EP 0 463 151 B1, grant published Jun. 12, 1996,International Patent Application No., WO 94/02602, published Feb. 3,1994, International Patent Application No., WO 96/34096, published Oct.31, 1996, WO 98/24893, published Jun. 11, 1998, WO 00/76310, publishedDec. 21, 2000. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilised a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (preferably a gamma constant region)are formed into a construct for insertion into an animal. This approachis described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat.Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference. Additionally, KM™—mice, which are theresult of cross-breeding of Kirin's Tc mice with Medarex's minilocus(Humab) mice have been generated. These mice possess the human IgHtranschromosome of the Kirin mice and the kappa chain transgene of theGenpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitableexamples include but are not limited to phage display (MedImmune,Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerlyProliferon), Affimed) ribosome display (MedImmune), yeast display, andthe like.

Preparation of Antibodies

Antibodies, as described herein, were prepared through the utilizationof the XenoMouse® technology, as described below. Such mice are capableof producing human immunoglobulin molecules and antibodies and aredeficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilised for achieving the same are disclosedin the patents, applications, and references disclosed in the backgroundsection herein. In particular, however, a preferred embodiment oftransgenic production of mice and antibodies therefrom is disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. See also Mendez et al. NatureGenetics 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

Through the use of such technology, fully human monoclonal antibodies toa variety of antigens have been produced. Essentially, XenoMouse® linesof mice are immunized with an antigen of interest (e.g. DLL4), lymphaticcells (such as B-cells) are recovered from the hyper-immunized mice, andthe recovered lymphocytes are fused with a myeloid-type cell line toprepare immortal hybridoma cell lines. These hybridoma cell lines arescreened and selected to identify hybridoma cell lines that producedantibodies specific to the antigen of interest. Provided herein aremethods for the production of multiple hybridoma cell lines that produceantibodies specific to DLL4. Further, provided herein arecharacterisation of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, B cells can be directly assayed. For example, CD19+ B cellscan be isolated from hyperimmune XenoMouse® mice and allowed toproliferate and differentiate into antibody-secreting plasma cells.Antibodies from the cell supernatants are then screened by, for example,ELISA, FACS or FMAT, for reactivity against the DLL4 immunogen. Thesupernatants might also be screened for immunoreactivity againstfragments of DLL4 to further map the different antibodies for binding todomains of functional interest on DLL4. The antibodies may also bescreened other related human DLL4s and against the rat, the mouse, andnon-human primate, such as Cynomolgus monkey, orthologues of DLL4, thelast to determine species cross-reactivity. B cells from wellscontaining antibodies of interest may be immortalised by various methodsincluding fusion to make hybridomas either from individual or frompooled wells, or by infection with EBV or transfection by knownimmortalising genes and then plating in suitable medium. Alternatively,single plasma cells secreting antibodies with the desired specificitiesare then isolated using an DLL4-specific hemolytic plaque assay (see forexample Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)).Cells targeted for lysis are preferably sheep red blood cells (SRBCs)coated with the DLL4 antigen.

In the presence of a B-cell culture containing plasma cells secretingthe immunoglobulin of interest and complement, the formation of a plaqueindicates specific DLL4-mediated lysis of the sheep red blood cellssurrounding the plasma cell of interest. The single antigen-specificplasma cell in the center of the plaque can be isolated and the geneticinformation that encodes the specificity of the antibody is isolatedfrom the single plasma cell. Using reverse-transcription followed by PCR(RT-PCR), the DNA encoding the heavy and light chain variable regions ofthe antibody can be cloned. Such cloned DNA can then be further insertedinto a suitable expression vector, preferably a vector cassette such asa pcDNA, more preferably such a pcDNA vector containing the constantdomains of immunglobulin heavy and light chain. The generated vector canthen be transfected into host cells, e.g., HEK293 cells, CHO cells, andcultured in conventional nutrient media modified as appropriate forinducing transcription, selecting transformants, or amplifying the genesencoding the desired sequences.

As will be appreciated, antibodies that specifically bind DLL4 can beexpressed in cell lines other than hybridoma cell lines. Sequencesencoding particular antibodies can be used to transform a suitablemammalian host cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216,4,912,040, 4,740,461, and 4,959,455 (which patents are herebyincorporated herein by reference). The transformation procedure useddepends upon the host to be transformed. Methods for introducingheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), human epithelial kidney 293 cells, and a number of othercell lines. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels and produceantibodies with constitutive DLL4 binding properties.

In the cell-cell fusion technique, a myeloma, CHO cell or other cellline is prepared that possesses a heavy chain with any desired isotypeand another myeloma, CHO cell or other cell line is prepared thatpossesses the light chain. Such cells can, thereafter, be fused and acell line expressing an intact antibody can be isolated.

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain of the desired “functional” attributesthrough isotype switching.

Therapeutic Administration and Formulations

Embodiments of the invention include sterile pharmaceutical formulationsof anti-DLL4 antibodies that are useful as treatments for diseases. Suchformulations would inhibit the binding of a native DLL4 to the Notch 1or Notch 4 receptor, thereby effectively treating pathologicalconditions where, for example, serum or tissue DLL4 expression isabnormally elevated. Anti-DLL4 antibodies preferably possess adequateaffinity to potently inhibit native DLL4 binding to the Notch 1 or Notch4 receptor and preferably have an adequate duration of action to allowfor infrequent dosing in humans. A prolonged duration of action willallow for less frequent and more convenient dosing schedules byalternate parenteral routes such as subcutaneous or intramuscularinjection.

Sterile formulations can be created, for example, by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution of the antibody. The antibody ordinarily will be storedin lyophilized form or in solution. Therapeutic antibody compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having an adapter thatallows retrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle.

The route of antibody administration is in accord with known methods,e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, direct injection to a tumor site, orby sustained release systems as noted below. The antibody is preferablyadministered continuously by infusion or by bolus injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it ispreferred that the therapist titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or by the assays describedherein.

Antibodies, as described herein, can be prepared in a mixture with apharmaceutically acceptable carrier. This therapeutic composition can beadministered intravenously or through the nose or lung, preferably as aliquid or powder aerosol (lyophilized). The composition may also beadministered parenterally or subcutaneously as desired. Whenadministered systemically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art. Briefly, dosage formulations of thecompounds described herein are prepared for storage or administration bymixing the compound having the desired degree of purity withpharmaceutically acceptable carriers, excipients, or stabilizers. Suchmaterials are non-toxic to the recipients at the dosages andconcentrations employed, and include buffers such as TRIS HCl,phosphate, citrate, acetate and other organic acid salts; antioxidantssuch as ascorbic acid; low molecular weight (less than about tenresidues) peptides such as polyarginine, proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidinone; amino acids such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington: TheScience and Practice of Pharmacy (20^(th) ed, Lippincott Williams &Wilkens Publishers (2003)). For example, dissolution or suspension ofthe active compound in a pharmaceutically acceptable carrier such aswater or naturally occurring vegetable oil like sesame, peanut, orcottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired. Buffers, preservatives, antioxidants and the likecan be incorporated according to accepted pharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed Mater. Res., (1981) 15:167-277 andLanger, Chem. Tech., (1982) 12:98-105, or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,(1983) 22:547-556), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-released compositions also include preparations of crystals ofthe antibody suspended in suitable formulations capable of maintainingcrystals in suspension. These preparations when injected subcutaneouslyor intraperitonealy can produce a sustained release effect. Othercompositions also include liposomally entrapped antibodies. Liposomescontaining such antibodies are prepared by methods known per se: U.S.Pat. No. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,(1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980)77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641;Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324.

The dosage of the antibody formulation for a given patient will bedetermined by the attending physician taking into consideration variousfactors known to modify the action of drugs including severity and typeof disease, body weight, sex, diet, time and route of administration,other medications and other relevant clinical factors. Therapeuticallyeffective dosages may be determined by either in vitro or in vivomethods.

An effective amount of the antibodies, described herein, to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it is preferred for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical daily dosage might range fromabout 0.0001 mg/kg, 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 10mg/kg to up to 100 mg/kg, 1000 mg/kg, 10000 mg/kg or more, of thepatient's body weight depending on the factors mentioned above. Thedosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg,0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's bodyweight depending on the factors mentioned above. Typically, theclinician will administer the therapeutic antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or as described herein.

Doses of antibodies of the invention may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months.

It will be appreciated that administration of therapeutic entities inaccordance with the compositions and methods herein will be administeredwith suitable carriers, excipients, and other agents that areincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilization and development of solid protein pharmaceuticals.” Int.J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs,and oral drug delivery-some emerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related toformulations, excipients and carriers well known to pharmaceuticalchemists.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto DLL4, the design of other therapeutic modalities beyond antibodymoieties is facilitated. Such modalities include, without limitation,advanced antibody therapeutics, such as bispecific antibodies,immunotoxins, and radiolabeled therapeutics, single domain antibodies,antibody fragments, such as a Fab, Fab′, F(ab′)₂, Fv or dAb, generationof peptide therapeutics, DLL4 binding domains in novel scaffolds, genetherapies, particularly intrabodies, antisense therapeutics, and smallmolecules.

An antigen binding site may be provided by means of arrangement of CDRson non-antibody protein scaffolds, such as fibronectin or cytochrome Betc. (Haan & Maggos (2004) BioCentury, 12(5): A1-A6; Koide et al. (1998)Journal of Molecular Biology, 284: 1141-1151; Nygren et al. (1997)Current Opinion in Structural Biology, 7: 463-469) or by randomising ormutating amino acid residues of a loop within a protein scaffold toconfer binding specificity for a desired target. Scaffolds forengineering novel binding sites in proteins have been reviewed in detailby Nygren et al. (Nygren et al. (1997) Current Opinion in StructuralBiology, 7: 463-469). Protein scaffolds for antibody mimics aredisclosed in WO/0034784, which is herein incorporated by reference inits entirety, in which the inventors describe proteins (antibody mimics)that include a fibronectin type III domain having at least onerandomised loop. A suitable scaffold into which to graft one or moreCDRs, e.g. a set of HCDRs, may be provided by any domain member of theimmunoglobulin gene superfamily. The scaffold may be a human ornon-human protein. An advantage of a non-antibody protein scaffold isthat it may provide an antigen-binding site in a scaffold molecule thatis smaller and/or easier to manufacture than at least some antibodymolecules. Small size of a binding member may confer usefulphysiological properties, such as an ability to enter cells, penetratedeep into tissues or reach targets within other structures, or to bindwithin protein cavities of the target antigen. Use of antigen bindingsites in non-antibody protein scaffolds is reviewed in Wess, 2004 (Wess,L. In: BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7,2004). Typical are proteins having a stable backbone and one or morevariable loops, in which the amino acid sequence of the loop or loops isspecifically or randomly mutated to create an antigen-binding site thatbinds the target antigen. Such proteins include the IgG-binding domainsof protein A from S. aureus, transferrin, albumin, tetranectin,fibronectin (e.g. 10th fibronectin type III domain), lipocalins as wellas gamma-crystalline and other Affilin™ scaffolds (Scil Proteins).Examples of other approaches include synthetic “Microbodies” based oncyclotides—small proteins having intra-molecular disulphide bonds,Microproteins (Versabodies™, Amunix) and ankyrin repeat proteins(DARPins, Molecular Partners).

In addition to antibody sequences and/or an antigen-binding site, atargeted binding agent according to the present invention may compriseother amino acids, e.g. forming a peptide or polypeptide, such as afolded domain, or to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. Targeted bindingagents of the invention may carry a detectable label, or may beconjugated to a toxin or a targeting moiety or enzyme (e.g. via apeptidyl bond or linker). For example, a targeted binding agent maycomprise a catalytic site (e.g. in an enzyme domain) as well as anantigen binding site, wherein the antigen binding site binds to theantigen and thus targets the catalytic site to the antigen. Thecatalytic site may inhibit biological function of the antigen, e.g. bycleavage.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it may be possibleto sidestep the dependence on complement for cell killing through theuse of bispecific antibodies, immunotoxins, or radiolabels, for example.

For example, bispecific antibodies can be generated that comprise (i)two antibodies one with a specificity to DLL4 and another to a secondmolecule that are conjugated together, (ii) a single antibody that hasone chain specific to DLL4 and a second chain specific to a secondmolecule, or (iii) a single chain antibody that has specificity to DLL4and the other molecule. Such bispecific antibodies can be generatedusing techniques that are well known; for example, in connection with(i) and (ii) see e.g., Fanger et al. Immunol Methods 4:72-81 (1994) andWright and Harris, supra. and in connection with (iii) see e.g.,Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992). In each case,the second specificity can be made to the heavy chain activationreceptors, including, without limitation, CD16 or CD64 (see e.g., Deo etal. Immunol. Today 18:127 (1997)) or CD89 (see e.g., Valerius et al.Blood 90:4485-4492 (1997)).

Antibodies can also be modified to act as immunotoxins, utilizingtechniques that are well known in the art. See e.g., Vitetta ImmunolToday 14:252 (1993). See also U.S. Pat. No. 5,194,594. In connectionwith the preparation of radiolabeled antibodies, such modifiedantibodies can also be readily prepared utilizing techniques that arewell known in the art. See e.g., Junghans et al. in Cancer Chemotherapyand Biotherapy 655-686 (2d edition, Chafner and Longo, eds., LippincottRaven (1996)). See also U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827,5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each immunotoxin orradiolabeled molecule would be likely to kill cells expressing thedesired multimeric enzyme subunit oligomerisation domain.

When an antibody is linked to an agent (e.g., radioisotope,pharmaceutical composition, or a toxin), it is contemplated that theagent possess a pharmaceutical property selected from the group ofantimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,alkaloid, COX-2, and antibiotic agents and combinations thereof. Thedrug can be selected from the group of nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs,purine analogs, antimetabolites, antibiotics, enzymes,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, antagonists, endostatin, taxols, camptothecins,oxaliplatin, doxorubicins and their analogs, and a combination thereof.

Examples of toxins further include gelonin, Pseudomonas exotoxin (PE),PE40, PE38, diphtheria toxin, ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, Pseudomonas endotoxin, members of theenediyne family of molecules, such as calicheamicin and esperamicin, aswell as derivatives, combinations and modifications thereof. Chemicaltoxins can also be taken from the group consisting of duocarmycin (see,e.g., U.S. Pat. No. 5,703,080 and U.S. Pat. No. 4,923,990),methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine,mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.Examples of chemotherapeutic agents also include Adriamycin,Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (Ara-C),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Pat. No.4,675,187), Melphalan, and other related nitrogen mustards. Suitabletoxins and chemotherapeutic agents are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and inGoodman And Gilman's The Pharmacological Basis of Therapeutics, 7th Ed.(MacMillan Publishing Co. 1985). Other suitable toxins and/orchemotherapeutic agents are known to those of skill in the art.

Examples of radioisotopes include gamma-emitters, positron-emitters, andx-ray emitters that can be used for localisation and/or therapy, andbeta-emitters and alpha-emitters that can be used for therapy. Theradioisotopes described previously as useful for diagnostics,prognostics and staging are also useful for therapeutics.

Non-limiting examples of anti-cancer or anti-leukemia agents includeanthracyclines such as doxorubicin (adriamycin), daunorubicin(daunomycin), idarubicin, detorubicin, caminomycin, epirubicin,esorubicin, and morpholino and substituted derivatives, combinations andmodifications thereof. Exemplary pharmaceutical agents includecis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C),cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine,chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide,and bleomycin, and derivatives, combinations and modifications thereof.Preferably, the anti-cancer or anti-leukemia is doxorubicin,morpholinodoxorubicin, or morpholinodaunorubicin.

The antibodies of the invention also encompass antibodies that havehalf-lives (e.g., serum half-lives) in a mammal, preferably a human, ofgreater than that of an unmodified antibody. Said antibody half life maybe greater than about 15 days, greater than about 20 days, greater thanabout 25 days, greater than about 30 days, greater than about 35 days,greater than about 40 days, greater than about 45 days, greater thanabout 2 months, greater than about 3 months, greater than about 4months, or greater than about 5 months. The increased half-lives of theantibodies of the present invention or fragments thereof in a mammal,preferably a human, result in a higher serum titer of said antibodies orantibody fragments in the mammal, and thus, reduce the frequency of theadministration of said antibodies or antibody fragments and/or reducesthe concentration of said antibodies or antibody fragments to beadministered. Antibodies or fragments thereof having increased in vivohalf-lives can be generated by techniques known to those of skill in theart. For example, antibodies or fragments thereof with increased in vivohalf-lives can be generated by modifying (e.g., substituting, deletingor adding) amino acid residues identified as involved in the interactionbetween the Fc domain and the FcRn receptor (see, e.g., InternationalPublication Nos. WO 97/34631 and WO 02/060919, which are incorporatedherein by reference in their entireties). Antibodies or fragmentsthereof with increased in vivo half-lives can be generated by attachingto said antibodies or antibody fragments polymer molecules such as highmolecular weight polyethyleneglycol (PEG). PEG can be attached to saidantibodies or antibody fragments with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of said antibodies or antibody fragments or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatisation that results in minimal loss of biological activity willbe used. The degree of conjugation will be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by, e.g., size exclusion or ion-exchange chromatography.

As will be appreciated by one of skill in the art, in the aboveembodiments, while affinity values can be important, other factors canbe as important or more so, depending upon the particular function ofthe antibody. For example, for an immunotoxin (toxin associated with anantibody), the act of binding of the antibody to the target can beuseful; however, in some embodiments, it is the internalisation of thetoxin into the cell that is the desired end result. As such, antibodieswith a high percent internalisation can be desirable in thesesituations. Thus, in one embodiment, antibodies with a high efficiencyin internalisation are contemplated. A high efficiency ofinternalisation can be measured as a percent internalised antibody, andcan be from a low value to 100%. For example, in varying embodiments,0.1-5, 5-10, 10-20, 20-30, 30-40, 40-45, 45-50, 50-60, 60-70, 70-80,80-90, 90-99, and 99-100% can be a high efficiency. As will beappreciated by one of skill in the art, the desirable efficiency can bedifferent in different embodiments, depending upon, for example, theassociated agent, the amount of antibody that can be administered to anarea, the side effects of the antibody-agent complex, the type (e.g.,cancer type) and severity of the problem to be treated.

In other embodiments, the antibodies disclosed herein provide an assaykit for the detection of DLL4 expression in mammalian tissues or cellsin order to screen for a disease or disorder associated with changes inexpression of DLL4. The kit comprises an antibody that binds DLL4 andmeans for indicating the reaction of the antibody with the antigen, ifpresent.

Combinations

The targeted binding agent or antibody defined herein may be applied asa sole therapy or may involve, in addition to the compounds of theinvention, conventional surgery or radiotherapy or chemotherapy. Suchchemotherapy may include one or more of the following categories of antitumor agents:

(i) other antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating agents (forexample cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); antitumorantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;

(iii) anti-invasion agents (for example c-Src kinase family inhibitorslike4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline(AZD0530; International Patent Application WO 01/94341) andN-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661), andmetalloproteinase inhibitors like marimastat, inhibitors of urokinaseplasminogen activator receptor function or, inhibitors of cathepsins,inhibitors of serine proteases for example matriptase, hepsin,urokinase, inhibitors of heparanase);

(iv) cytotoxic agents such as fludarabine, 2-chlorodeoxyadenosine,chlorambucil or doxorubicin and combination thereoff such asFludarabine+cyclophosphamide, CVP:cyclophosphamide+vincristine+prednisone, ACVBP:doxorubicin+cyclophosphamide+vindesine+bleomycin+prednisone, CHOP:cyclophosphamide+doxorubicin+vincristine+prednisone, CNOP:cyclophosphamide+mitoxantrone+vincristine+prednisone, m-BACOD:methotrexate+bleomycin+doxorubicin+cyclophosphamide+vincristine+dexamethasone+leucovorin,MACOP-B:methotrexate+doxorubicin+cyclophosphamide+vincristine+prednisone fixeddose+bleomycin+leucovorin, or ProMACE CytaBOM:prednisone+doxorubicin+cyclophosphamide+etoposide+cytarabine+bleomycin+vincristine+methotrexate+leucovorin.

(v) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab[Erbitux, C225] and any growth factor or growth factor receptorantibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors alsoinclude tyrosine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, ZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib,inhibitors of the hepatocyte growth factor family, inhibitors of theplatelet-derived growth factor family such as imatinib, inhibitors ofserine/threonine kinases (for example Ras/Raf signalling inhibitors suchas farnesyl transferase inhibitors, for example sorafenib (BAY43-9006)), inhibitors of cell signalling through MEK and/or AKT kinases,inhibitors of the hepatocyte growth factor family, c-kit inhibitors, ablkinase inhibitors, IGF receptor (insulin-like growth factor) kinaseinhibitors, aurora kinase inhibitors (for example AZD1152, PH739358,VX-680, MLN8054, R763, MP235, MP529, VX-528 and AX39459), cyclindependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors, andinhibitors of survival signaling proteins such as Bcl-2, Bcl-XL forexample ABT-737;

(vi) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, [for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™),Angiopoietin-2 inhibitors, and VEGF receptor tyrosine kinase inhibitorssuch as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814; Sutent™), Sorafenib(Nexxavar™), compounds such as those disclosed in International PatentApplications WO97/22596, WO 97/30035, WO 97/32856, WO 98/13354,WO00/47212 and WO01/32651 and compounds that work by other mechanisms(for example linomide, inhibitors of integrin αvβ3 function andangiostatin)] or colony stimulating factor 1 (CSF1) or CSF1 receptor;

(vii) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(viii) antisense therapies, for example those which are directed to thetargets listed above, such as G-3139 (Genasense), an anti blc2antisense;

(ix) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene directed enzyme pro drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi drug resistance gene therapy; and

(x) immunotherapy approaches, including for example treatment withAlemtuzumab (campath-1H™), a monoclonal antibody directed at CD52, ortreatment with antibodies directed at CD22, ex vivo and in vivoapproaches to increase the immunogenicity of patient tumor cells,transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte macrophage colony stimulating factor, approaches to decreaseT cell anergy such as treatment with monoclonal antibodies inhibitingCTLA-4 function, approaches using transfected immune cells such ascytokine transfected dendritic cells, approaches using cytokinetransfected tumor cell lines and approaches using anti idiotypicantibodies.

(xi) inhibitors of protein degradation such as proteasome inhibitor suchas Velcade (bortezomib).

(xii) biotherapeutic therapeutic approaches for example those which usepeptides or proteins (such as antibodies or soluble external receptordomain constructions) which either sequester receptor ligands, blockligand binding to receptor or decrease receptor signalling (e.g. due toenhanced receptor degradation or lowered expression levels).

In one embodiment the anti-tumor treatment defined herein may involve,in addition to the compounds of the invention, treatment with otherantiproliferative/antineoplastic drugs and combinations thereof, as usedin medical oncology, such as alkylating agents (for example cis-platin,oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites(for example gemcitabine and antifolates such as fluoropyrimidines like5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosinearabinoside, and hydroxyurea); antitumor antibiotics (for exampleanthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin,epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin);antimitotic agents (for example vinca alkaloids like vincristine,vinblastine, vindesine and vinorelbine and taxoids like taxol andtaxotere and polokinase inhibitors); and topoisomerase inhibitors (forexample epipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan and camptothecin).

In one embodiment the anti-tumor treatment defined herein may involve,in addition to the compounds of the invention, treatment withgemcitabine.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such combination products employ the compounds of thisinvention, or pharmaceutically acceptable salts thereof, within thedosage range described hereinbefore and the other pharmaceuticallyactive agent within its approved dosage range.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the teachings herein.

Example 1 Immunization and Titering Immunogens

The extracellular domain of human DLL4 (amino acids 1-524) andrecombinant human DLL4 transiently expressed in Chinese Hamster Ovary(CHO) cells were used as antigens for immunizations. For the generationof the CHO transfectants, human full length DLL4 cDNA (Yoneya et al.,2001, J. Biochem., 129, 27-34) was inserted into pcDNA3.1 vector andlipofected into CHO cells (American Type Tissue Collection, catalog#CCL-61). Expression of human DLL4 at the cell surface at the levelsuitable for the purpose of immunization was confirmed by fluorescentactivated cell sorting (FACS) analysis. The extracellular domain ofhuman DLL4 was subcloned from full length DLL4 using the forward5′-AAGCTGGCTAGCGCGAATGGCGGCAGCGTCCCGGAG (SEQ ID NO: 92) and reverse5′-CAGCCTCGAGCGGCCGCCCAGGGGAAGCTGGGCGGCAAGC (SEQ ID NO: 93) primers. ThePCR product was purified and ligated into the pSecTag expression vectorfrom Invitrogen. The clone was subsequently transfected into 293T cellsusing 293fectin transfection reagent. After 7 days, the cellsupernatants containing the target protein were harvested and run over apre-equilibrated HisTrap column (GE Healthcare, catalog #17-5247)overnight. The column was washed with a binding buffer containing 20 mMsodium phosphate, 500 mM sodium chloride and 5 mM imidazole, pH 7.4before the his-tagged protein was eluted in a buffer containing 20 mMsodium phosphate, 500 mM sodium chloride and 500 mM imidazole, pH 7.4.The protein sample was dialyzed in binding buffer for 1 h at 4° C.before being further dialyzed in PBS, pH 7.4 for 2 h prior to filtersterilization, quantification and purity assessment by SDS-PAGE followedby staining with Gelcode (Pierce, catalog #24950).

Immunization

Monoclonal antibodies against DLL4 were developed by sequentiallyimmunizing XenoMouse® mice (XenoMouse strains: XMG2 (IgG2 kappa/lambda)and XMG4 (IgG4 kappa/lambda) Amgen, Inc. Vancouver, British Columbia,Canada) with either the extracellular domain of DLL4 or CHO cellsoverexpressing recombinant human DLL4 as described in Example 1.XenoMouse animals were immunized via intraperitoneal and base of tailroutes for all injections by conventional means. Adjuvants includedTitermax Gold™ (Sigma, catalog #T2684), aluminum phosphate gel adjuvant,HCL Biosector, (catalog #1452-250) and ImmuneEasy mouse adjuvant (qCpG,Qiagen catalog #303105). For the soluble immunogen, the first injectionof 10 μg DLL4 extracellular domain was administered with Titermax Gold™(Day 0) and alternate boosts were administered using either aluminumphosphate gel adjuvant and qCpG or Titremax Gold™ along with 5 μl ofDLL4 extracellular domain. For the cell-based immunogen, the firstinjection was administered with aluminum phosphate gel adjuvant and 2E6cells. During subsequent boosts 1E6 cells were administered using thesame adjuvant. For both immunization campaigns, boosting occurred ondays 2, 6, 10, 16, 23, 30, 37, 44, 50, 64, 71, 75, 89, 104 and 108.

Selections of Animals for Harvest by Titer

Titers of the antibodies against human DLL4 were tested by for bindingto human and mouse DLL4 expressed in 293T cells using a Fluorometricmicrovolume assay technology (FMAT) cellular detection instrument(Applied Biosystems). This analysis showed that there were some micethat had titers, which appeared to be specific for DLL4. Therefore, atthe end of the immunization programme, 17 mice were selected forharvest, and lymphocytes were isolated from the spleens and lymph nodesof the immunized mice as described in example 2 below.

Example 2 Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

Immunized mice were sacrificed by cervical dislocation and the draininglymph nodes were harvested and pooled from each cohort. The lymphoidcells were dissociated by grinding in DMEM to release the cells from thetissues, and the cells were suspended in DMEM. B cells were enriched bypositive selection using CD19 labelled Dynal beads. A fusion wasperformed by mixing washed enriched B cells from above and nonsecretorymyeloma P3X63Ag8.653 cells purchased from ATCC (catalog #CRL 1580)(Kearney et al., J. Immunol. 123, 1979, 1548-1550) at a ratio of 1:1.The cell mixture was gently pelleted by centrifugation at 800×g. Aftercomplete removal of the supernatant, the cells were treated with 2-4 mlof Pronase solution (CalBiochem, catalog #53702; 0.5 mg/ml in PBS) forno more than 2 minutes. Then 3-5 ml of FBS was added to stop the enzymeactivity and the suspension was adjusted to 40 ml total volume usingelectro cell fusion solution, ECFS (0.3 M sucrose, Sigma, catalog#S7903, 0.1 mM magnesium acetate, Sigma, catalog #M2545, 0.1 mM calciumacetate, Sigma, catalog #C4705). The supernatant was removed aftercentrifugation and the cells were resuspended in 40 ml ECFS. This washstep was repeated and the cells again were resuspended in ECFS to aconcentration of 2E6 cells/ml. Electro-cell fusion was performed using afusion generator, model ECM2001, Genetronic, Inc., San Diego, Calif. Thefusion chamber size used was 2.0 ml, using the following instrumentsettings: alignment condition: voltage: 50 V, time: 50 seconds; membranebreaking at: voltage: 3000 V, time: 30 μseconds; post-fusion holdingtime: 3 seconds. After ECF, the cell suspensions were carefully removedfrom the fusion chamber under sterile conditions and transferred into asterile tube containing the same volume of Hybridoma Culture Medium(DMEM (JRH Biosciences), 15% FBS (Hyclone), supplemented with 2 mML-glutamine (Sigma, catalog #G2150), 10 U/ml penicillin/0.1 mg/mlstreptomycin (Sigma, catalog #P7539), 1 vial/L OPI (oxaloacetate,pyruvate, bovine insulin; Sigma catalog #O5003) and 10 U/ml recombinanthuman IL-6 (Boehringer Mannheim, catalog #1131567). The cells wereincubated for 15-30 minutes at 37° C., and then centrifuged at 400×g for5 min. The cells were gently resuspended in a small volume of HybridomaSelection Medium (Hybridoma Culture Medium supplemented with 0.5×HA(Sigma, catalog #A9666)), and the volume was adjusted appropriately withmore Hybridoma Selection Medium, based on a final plating of 5E6 B cellstotal per 96-well plate and 200 μl per well. The cells were mixed gentlyand pipetted into 96-well plates and allowed to grow. Exhaustivesupernatants were collected from the cells that potentially produceanti-DLL4 antibodies and subjected to subsequent screening assays asexemplified below.

Example 3 Binding to Cell Bound Human and Cynomolgus Monkey Dll4 andHuman Jagged1

Supernatants collected from harvested cells were tested to assess theability of the secreted antibodies to bind to 293T cells transientlyoverexpressing either full length human or cynomolgus monkey DLL4 orhuman Jagged1. A mock-transfected 293T cell line was used as a negativecontrol. Cells diluted in PBS containing 2% FBS were seeded at a densityof 3000 expressing and 15000 mock transfected cells per well in 384 wellplates (Corning Costar, catalog #3712). Immediately after plating, 15 or20 μl/well of hybridoma supernatants and 10 μl/well of secondaryantibody (Goat anti-human IgG Fc Cy5, final concentration 750 ng/ml)were added and plates incubated for 3 h at room temperature prior toreading the fluoresence on the FMAT 8200 instrument (AppliedBiosystems). The binding of human Notch1/Fc chimera (R&D systems,catalog #3647-TK), diluted 1:2 from 2.86 μg/ml was used as a positivecontrol for DLL4 and human Notch3/Fc chimera diluted from 10 μg/ml wasused a positive control for binding to Jagged1. Results for 12 hybridomasupernatants showing binding of hybridoma supernatants tohuman/cynomolgus monkey DLL4 and human Jagged are shown in Table 3.

TABLE 3 Cynomolgus monkey Antibody Human DLL4 binding DLL4 binding HumanJagged1 binding ID Count FL1 FL1 × count Count FL1 FL1 × count Count FL1FL1 × count 1D4 196 10600 2.08E6 103 11700 1.21E6 23 3250 74800 1E4 20610400 2.14E6 107 13200 1.41E6 2 8020 16000 4B4 194 9930 1.93E6 117 123001044E6  2 2590 5180 2H10 191 10800 2.06E6 103 13100 1.35E6 5 5030 252003A7 166 9470 1.57E6 121 9960 1.20E6 1 1940 1940 4B3 206 11000 2.27E6 9712600 1.22E6 2 4720 9450 9G8 198 9810 1.94E6 120 11700 1.41E6 0 0 0 12A1179 9520 1.76E6 106 12900 1.37E6 25 988 24700 17F3 219 11300 2.47E6 12010700 1.29E6 30 1440 43100 21F7 200 10400 2.08E6 103 12500 1.28E6 1 20702070 20G8 182 10500 1.90E6 128 11200 1.44E6 3 1500 4490 21H3 181 105001.91E6 120 11700 1.40E6 0 0 0

Example 4 Inhibition of Notch1-DLL4 Receptor-Ligand Binding

In order to determine the relative potency of the antibody containingsupernatants, their ability to inhibit the binding of human Notch1/Fc tohuman DLL4 transiently overexpressed in 293T cells was evaluated.Transfected and untransfected 239T cells were reconstituted in PBScontaining 2% FCS and 5000 transfected and 17500 non-transfected cellswere plated in 20 μl into wells of a 384-well tissue culture plate(Corning Costar, catalog #3712). Subsequently, 20 μl of hybridomasupernatant was added and plates were incubated at 4° C. for 1 h, atwhich time 20 μl of Alexa-647 labeled human Notch1/Fc was added at afinal concentration of 6.7 ng/ml. After a further 3 h incubation at 4°C., the amount of bound Notch1/Fc was determined by reading thefluorescence in each well using an FMAT 8200 instrument (AppliedBiosystems). The results for 12 hybridoma supernatants are shown inTable 4. Results are expressed as % inhibitions with the minimuminhibition in the assay being determined by the effects of non-DLL4binding supernatants prepared in a similar way as described in example2, and the maximum inhibition being defined as the signal obtained inthe presence of a saturating concentration of unlabeled Notch1/Fc.N.T.=not tested

TABLE 4 % inhibition % inhibition % inhibition Mean % Antibody ID n = 1n = 2 n = 3 inhibition 1D4 105 71 146 107 1E4 98 99 113 110 4B4 105 109148 121 2H10 107 150 124 127 3A7 105 120 142 122 4B3 106 145 147 133 9G8112 98 143 118 12A1 103 147 144 131 17F3 N.T. 108 131 120 21F7 N.T. 93140 117 20G8 N.T. 88 143 116 21H3 N.T. 131 140 136

Example 5 Structural Analysis of Anti-DLL4 Antibodies

The variable heavy chains and the variable light chains of theantibodies were sequenced to determine their DNA sequences. The completesequence information for the anti-DLL4 antibodies is provided in thesequence listing with nucleotide and amino acid sequences for each gammaand kappa chain combination. The variable heavy sequences were analyzedto determine the VH family, the D-region sequence and the J-regionsequence. The sequences were then translated to determine the primaryamino acid sequence and compared to the germline VH, D and J-regionsequences to assess somatic hypermutations.

Table 2 is a table comparing the antibody heavy chain regions to theircognate germ line heavy chain region and kappa light chain regions totheir cognate germ line lightchain region. The variable (V) regions ofimmunoglobulin chains are encoded by multiple germ line DNA segments,which are joined into functional variable regions (V_(H)DJ_(H) orV_(K)J_(K)) during B-cell ontogeny. The molecular and genetic diversityof the antibody response to DLL4 was studied in detail.

It should also be appreciated that where a particular antibody differsfrom its respective germline sequence at the amino acid level, theantibody sequence can be mutated back to the germline sequence. Suchcorrective mutations can occur at one, two, three or more positions, ora combination of any of the mutated positions, using standard molecularbiological techniques. By way of non-limiting example, Table 8 showsthat the light chain sequence of 2H10 (SEQ ID NO.: 6) differs from thecorresponding germline sequence (see Table 2) through a V to an A atposition 18 (mutation 1), a V to an A at position 32 (mutation 2), an Eto a D at position 50 (mutation 3), an S to an N at position 65(mutation 4), a T to an A at position 89 (mutation 5) and an L to a T atposition 94 (mutation 6). Thus, the amino acid or nucleotide sequenceencoding the light chain of 2H10 can be modified at any or all of thesesites. Tables 2-9 below illustrate the positions of such variations fromthe germline for 2H10, 9G8, 21H3 and 4B4. Each row represents a uniquecombination of germline and non-germline residues at the positionindicated by bold type.

In another embodiment, the invention includes replacing any structuralliabilities in the sequence that might affect the heterogeneity of theantibodies of the invention. Such liabilities include glycosylationsites, un-paired cysteines, surface exposed methinones, etc. To reducethe risk of such heterogeneity it is proposed that changes are made toremove one or more of such structural liabilities.

In one example, unpaired cysteines can be replaced alone or inconjunction with other structural changes. An example of an unpairedcysteine occurs in the light chain CDR1 of antibody 2H10 or 9G8 atposition 33. This unpaired cysteine can be mutated to an appropriateamino acid that has comparable side chain property such as a serine. Inanother example, an unpaired cysteine occurs in the heavy chain FR4 ofantibody 20G8 at position 203. This unpaired cysteine can likewise bemutated to an appropriate amino acid that has comparable side chainproperties such as a serine.

TABLE 5 Exemplary Mutations of 21H3 Heavy Chain (SEQ ID NO: 30) toGermline at the Indicated Residue Number 31 35 45 66 70 100 N T P G V RS T P G V R N S P G V R S S P G V R N T L G V R S T L G V R N S L G V RS S L G V R N T P D V R S T P D V R N S P D V R S S P D V R N T L D V RS T L D V R N S L D V R S S L D V R N T P G M R S T P G M R N S P G M RS S P G M R N T L G M R S T L G M R N S L G M R S S L G M R N T P D M RS T P D M R N S P D M R S S P D M R N T L D M R S T L D M R N S L D M RS S L D M R N T P G V I S T P G V I N S P G V I S S P G V I N T L G V IS T L G V I N S L G V I S S L G V I N T P D V I S T P D V I N S P D V IS S P D V I N T L D V I S T L D V I N S L D V I S S L D V I N T P G M IS T P G M I N S P G M I S S P G M I N T L G M I S T L G M I N S L G M IS S L G M I N T P D M I S T P D M I N S P D M I S S P D M I N T L D M IS T L D M I N S L D M I S S L D M I

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 30. In certainembodiments, SEQ ID NO.: 30 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 5. Insome embodiments, SEQ ID NO: 30 comprises any one, any two, any three,any four, any five, any six, or all six of the germline residues asindicated in Table 5. In certain embodiments, SEQ ID NO.: 30 comprisesany one of the unique combinations of germline and non-germline residuesindicated by each row of Table 5. In other embodiments, the targetedbinding agent or antibody is derived from a germline sequence withVH1-18, D2-15 and JH3 domains, wherein one or more residues has beenmutated to yield the corresponding germline residue at that position.Specific examples of 21H3 variable heavy domain which has been mutatedto particular germline sequences include 21H3VHOP (optimized where thenon-germline sequence has been mutated to an L at position 45 and an Mat position 70) as shown in Table 13.

TABLE 6 Exemplary Mutations of 21H3 Light Chain (SEQ ID NO: 32) toGermline at the Indicated Residue Number 32 33 67 99 107 Y F E H R N F EH R Y Y E H R N Y E H R Y F K H R N F K H R Y Y K H R N Y K H R Y F E VR N F E V R Y Y E V R N Y E V R Y F K V R N F K V R Y Y K V R N Y K V RY F E H K N F E H K Y Y E H K N Y E H K Y F K H K N F K H K Y Y K H K NY K H K Y F E V K N F E V K Y Y E V K N Y E V K Y F K V K N F K V K Y YK V K N Y K V K

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 32. In certainembodiments, SEQ ID NO.: 32 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 6. Insome embodiments, SEQ ID NO: 32 comprises any one, any two, any three,any four, any five, or all five of the germline residues as indicated inTable 6. In other embodiments, the targeted binding agent or antibody isderived from a germline sequence with VL, 1g, JL2 domains, wherein oneor more residues has been mutated to yield the corresponding germlineresidue at that position. Specific examples of 21H3 variable lightdomain which has been mutated to particular germline sequences include21H3 VLOP1 (optimized where the non-germline sequence has been mutatedto a K at position 107) and 21H3 VLOP2 (optimized where the non-germlinesequence has been mutated to a K at positions 67 and 107). See Table 13.

TABLE 7 Exemplary Mutations of 4B4 Heavy Chain (SEQ ID NO: 2) toGermline at the Indicated Residue Number 3 5 19 31 34 35 47 66 72 77 103L I Q N V I Y D S T S Q I Q N V I Y D S T S L V Q N V I Y D S T S Q V QN V I Y D S T S L I K N V I Y D S T S Q I K N V I Y D S T S L V K N V IY D S T S Q V K N V I Y D S T S L I Q S V I Y D S T S Q I Q S V I Y D ST S L V Q S V I Y D S T S Q V Q S V I Y D S T S L I K S V I Y D S T S QI K S V I Y D S T S L V K S V I Y D S T S Q V K S V I Y D S T S L I Q NI I Y D S T S Q I Q N I I Y D S T S L V Q N I I Y D S T S Q V Q N I I YD S T S L I K N I I Y D S T S Q I K N I I Y D S T S L V K N I I Y D S TS Q V K N I I Y D S T S L I Q S I I Y D S T S Q I Q S I I Y D S T S L VQ S I I Y D S T S Q V Q S I I Y D S T S L I K S I I Y D S T S Q I K S II Y D S T S L V K S I I Y D S T S Q V K S I I Y D S T S L I Q N V S Y DS T S Q I Q N V S Y D S T S L V Q N V S Y D S T S Q V Q N V S Y D S T SL I K N V S Y D S T S Q I K N V S Y D S T S L V K N V S Y D S T S Q V KN V S Y D S T S L I Q S V S Y D S T S Q I Q S V S Y D S T S L V Q S V SY D S T S Q V Q S V S Y D S T S L I K S V S Y D S T S Q I K S V S Y D ST S L V K S V S Y D S T S Q V K S V S Y D S T S L I Q N I S Y D S T S QI Q N I S Y D S T S L V Q N I S Y D S T S Q V Q N I S Y D S T S L I K NI S Y D S T S Q I K N I S Y D S T S L V K N I S Y D S T S Q V K N I S YD S T S L I Q S I S Y D S T S Q I Q S I S Y D S T S L V Q S I S Y D S TS Q V Q S I S Y D S T S L I K S I S Y D S T S Q I K S I S Y D S T S L VK S I S Y D S T S Q V K S I S Y D S T S L I Q N V I W D S T S Q I Q N VI W D S T S L V Q N V I W D S T S Q V Q N V I W D S T S L I K N V I W DS T S Q I K N V I W D S T S L V K N V I W D S T S Q V K N V I W D S T SL I Q S V I W D S T S Q I Q S V I W D S T S L V Q S V I W D S T S Q V QS V I W D S T S L I K S V I W D S T S Q I K S V I W D S T S L V K S V IW D S T S Q V K S V I W D S T S L I Q N I I W D S T S Q I Q N I I W D ST S L V Q N I I W D S T S Q V Q N I I W D S T S L I K N I I W D S T S QI K N I I W D S T S L V K N I I W D S T S Q V K N I I W D S T S L I Q SI I W D S T S Q I Q S I I W D S T S L V Q S I I W D S T S Q V Q S I I WD S T S L I K S I I W D S T S Q I K S I I W D S T S L V K S I I W D S TS Q V K S I I W D S T S L I Q N V S W D S T S Q I Q N V S W D S T S L VQ N V S W D S T S Q V Q N V S W D S T S L I K N V S W D S T S Q I K N VS W D S T S L V K N V S W D S T S Q V K N V S W D S T S L I Q S V S W DS T S Q I Q S V S W D S T S L V Q S V S W D S T S Q V Q S V S W D S T SL I K S V S W D S T S Q I K S V S W D S T S L V K S V S W D S T S Q V KS V S W D S T S L I Q N I S W D S T S Q I Q N I S W D S T S L V Q N I SW D S T S Q V Q N I S W D S T S L I K N I S W D S T S Q I K N I S W D ST S L V K N I S W D S T S Q V K N I S W D S T S L I Q S I S W D S T S QI Q S I S W D S T S L V Q S I S W D S T S Q V Q S I S W D S T S L I K SI S W D S T S Q I K S I S W D S T S L V K S I S W D S T S Q V K S I S WD S T S L I Q N V I Y G S T S Q I Q N V I Y G S T S L V Q N V I Y G S TS Q V Q N V I Y G S T S L I K N V I Y G S T S Q I K N V I Y G S T S L VK N V I Y G S T S Q V K N V I Y G S T S L I Q S V I Y G S T S Q I Q S VI Y G S T S L V Q S V I Y G S T S Q V Q S V I Y G S T S L I K S V I Y GS T S Q I K S V I Y G S T S L V K S V I Y G S T S Q V K S V I Y G S T SL I Q N I I Y G S T S Q I Q N I I Y G S T S L V Q N I I Y G S T S Q V QN I I Y G S T S L I K N I I Y G S T S Q I K N I I Y G S T S L V K N I IY G S T S Q V K N I I Y G S T S L I Q S I I Y G S T S Q I Q S I I Y G ST S L V Q S I I Y G S T S Q V Q S I I Y G S T S L I K S I I Y G S T S QI K S I I Y G S T S L V K S I I Y G S T S Q V K S I I Y G S T S L I Q NV S Y G S T S Q I Q N V S Y G S T S L V Q N V S Y G S T S Q V Q N V S YG S T S L I K N V S Y G S T S Q I K N V S Y G S T S L V K N V S Y G S TS Q V K N V S Y G S T S L I Q S V S Y G S T S Q I Q S V S Y G S T S L VQ S V S Y G S T S Q V Q S V S Y G S T S L I K S V S Y G S T S Q I K S VS Y G S T S L V K S V S Y G S T S Q V K S V S Y G S T S L I Q N I S Y GS T S Q I Q N I S Y G S T S L V Q N I S Y G S T S Q V Q N I S Y G S T SL I K N I S Y G S T S Q I K N I S Y G S T S L V K N I S Y G S T S Q V KN I S Y G S T S L I Q S I S Y G S T S Q I Q S I S Y G S T S L V Q S I SY G S T S Q V Q S I S Y G S T S L I K S I S Y G S T S Q I K S I S Y G ST S L V K S I S Y G S T S Q V K S I S Y G S T S L I Q N V I W G S T S QI Q N V I W G S T S L V Q N V I W G S T S Q V Q N V I W G S T S L I K NV I W G S T S Q I K N V I W G S T S L V K N V I W G S T S Q V K N V I WG S T S L I Q S V I W G S T S Q I Q S V I W G S T S L V Q S V I W G S TS Q V Q S V I W G S T S L I K S V I W G S T S Q I K S V I W G S T S L VK S V I W G S T S Q V K S V I W G S T S L I Q N I I W G S T S Q I Q N II W G S T S L V Q N I I W G S T S Q V Q N I I W G S T S L I K N I I W GS T S Q I K N I I W G S T S L V K N I I W G S T S Q V K N I I W G S T SL I Q S I I W G S T S Q I Q S I I W G S T S L V Q S I I W G S T S Q V QS I I W G S T S L I K S I I W G S T S Q I K S I I W G S T S L V K S I IW G S T S Q V K S I I W G S T S L I Q N V S W G S T S Q I Q N V S W G ST S L V Q N V S W G S T S Q V Q N V S W G S T S L I K N V S W G S T S QI K N V S W G S T S L V K N V S W G S T S Q V K N V S W G S T S L I Q SV S W G S T S Q I Q S V S W G S T S L V Q S V S W G S T S Q V Q S V S WG S T S L I K S V S W G S T S Q I K S V S W G S T S L V K S V S W G S TS Q V K S V S W G S T S L I Q N I S W G S T S Q I Q N I S W G S T S L VQ N I S W G S T S Q V Q N I S W G S T S L I K N I S W G S T S Q I K N IS W G S T S L V K N I S W G S T S Q V K N I S W G S T S L I Q S I S W GS T S Q I Q S I S W G S T S L V Q S I S W G S T S Q V Q S I S W G S T SL I K S I S W G S T S Q I K S I S W G S T S L V K S I S W G S T S Q V KS I S W G S T S L I Q N V I Y D T T S Q I Q N V I Y D T T S L V Q N V IY D T T S Q V Q N V I Y D T T S L I K N V I Y D T T S Q I K N V I Y D TT S L V K N V I Y D T T S Q V K N V I Y D T T S L I Q S V I Y D T T S QI Q S V I Y D T T S L V Q S V I Y D T T S Q V Q S V I Y D T T S L I K SV I Y D T T S Q I K S V I Y D T T S L V K S V I Y D T T S Q V K S V I YD T T S L I Q N I I Y D T T S Q I Q N I I Y D T T S L V Q N I I Y D T TS Q V Q N I I Y D T T S L I K N I I Y D T T S Q I K N I I Y D T T S L VK N I I Y D T T S Q V K N I I Y D T T S L I Q S I I Y D T T S Q I Q S II Y D T T S L V Q S I I Y D T T S Q V Q S I I Y D T T S L I K S I I Y DT T S Q I K S I I Y D T T S L V K S I I Y D T T S Q V K S I I Y D T T SL I Q N V S Y D T T S Q I Q N V S Y D T T S L V Q N V S Y D T T S Q V QN V S Y D T T S L I K N V S Y D T T S Q I K N V S Y D T T S L V K N V SY D T T S Q V K N V S Y D T T S L I Q S V S Y D T T S Q I Q S V S Y D TT S L V Q S V S Y D T T S Q V Q S V S Y D T T S L I K S V S Y D T T S QI K S V S Y D T T S L V K S V S Y D T T S Q V K S V S Y D T T S L I Q NI S Y D T T S Q I Q N I S Y D T T S L V Q N I S Y D T T S Q V Q N I S YD T T S L I K N I S Y D T T S Q I K N I S Y D T T S L V K N I S Y D T TS Q V K N I S Y D T T S L I Q S I S Y D T T S Q I Q S I S Y D T T S L VQ S I S Y D T T S Q V Q S I S Y D T T S L I K S I S Y D T T S Q I K S IS Y D T T S L V K S I S Y D T T S Q V K S I S Y D T T S L I Q N V I W DT T S Q I Q N V I W D T T S L V Q N V I W D T T S Q V Q N V I W D T T SL I K N V I W D T T S Q I K N V I W D T T S L V K N V I W D T T S Q V KN V I W D T T S L I Q S V I W D T T S Q I Q S V I W D T T S L V Q S V IW D T T S Q V Q S V I W D T T S L I K S V I W D T T S Q I K S V I W D TT S L V K S V I W D T T S Q V K S V I W D T T S L I Q N I I W D T T S QI Q N I I W D T T S L V Q N I I W D T T S Q V Q N I I W D T T S L I K NI I W D T T S Q I K N I I W D T T S L V K N I I W D T T S Q V K N I I WD T T S L I Q S I I W D T T S Q I Q S I I W D T T S L V Q S I I W D T TS Q V Q S I I W D T T S L I K S I I W D T T S Q I K S I I W D T T S L VK S I I W D T T S Q V K S I I W D T T S L I Q N V S W D T T S Q I Q N VS W D T T S L V Q N V S W D T T S Q V Q N V S W D T T S L I K N V S W DT T S Q I K N V S W D T T S L V K N V S W D T T S Q V K N V S W D T T SL I Q S V S W D T T S Q I Q S V S W D T T S L V Q S V S W D T T S Q V QS V S W D T T S L I K S V S W D T T S Q I K S V S W D T T S L V K S V SW D T T S Q V K S V S W D T T S L I Q N I S W D T T S Q I Q N I S W D TT S L V Q N I S W D T T S Q V Q N I S W D T T S L I K N I S W D T T S QI K N I S W D T T S L V K N I S W D T T S Q V K N I S W D T T S L I Q SI S W D T T S Q I Q S I S W D T T S L V Q S I S W D T T S Q V Q S I S WD T T S L I K S I S W D T T S Q I K S I S W D T T S L V K S I S W D T TS Q V K S I S W D T T S L I Q N V I Y G T T S Q I Q N V I Y G T T S L VQ N V I Y G T T S Q V Q N V I Y G T T S L I K N V I Y G T T S Q I K N VI Y G T T S L V K N V I Y G T T S Q V K N V I Y G T T S L I Q S V I Y GT T S Q I Q S V I Y G T T S L V Q S V I Y G T T S Q V Q S V I Y G T T SL I K S V I Y G T T S Q I K S V I Y G T T S L V K S V I Y G T T S Q V KS V I Y G T T S L I Q N I I Y G T T S Q I Q N I I Y G T T S L V Q N I IY G T T S Q V Q N I I Y G T T S L I K N I I Y G T T S Q I K N I I Y G TT S L V K N I I Y G T T S Q V K N I I Y G T T S L I Q S I I Y G T T S QI Q S I I Y G T T S L V Q S I I Y G T T S Q V Q S I I Y G T T S L I K SI I Y G T T S Q I K S I I Y G T T S L V K S I I Y G T T S Q V K S I I YG T T S L I Q N V S Y G T T S Q I Q N V S Y G T T S L V Q N V S Y G T TS Q V Q N V S Y G T T S L I K N V S Y G T T S Q I K N V S Y G T T S L VK N V S Y G T T S Q V K N V S Y G T T S L I Q S V S Y G T T S Q I Q S VS Y G T T S L V Q S V S Y G T T S Q V Q S V S Y G T T S L I K S V S Y GT T S Q I K S V S Y G T T S L V K S V S Y G T T S Q V K S V S Y G T T SL I Q N I S Y G T T S Q I Q N I S Y G T T S L V Q N I S Y G T T S Q V QN I S Y G T T S L I K N I S Y G T T S Q I K N I S Y G T T S L V K N I SY G T T S Q V K N I S Y G T T S L I Q S I S Y G T T S Q I Q S I S Y G TT S L V Q S I S Y G T T S Q V Q S I S Y G T T S L I K S I S Y G T T S QI K S I S Y G T T S L V K S I S Y G T T S Q V K S I S Y G T T S L I Q NV I W G T T S Q I Q N V I W G T T S L V Q N V I W G T T S Q V Q N V I WG T T S L I K N V I W G T T S Q I K N V I W G T T S L V K N V I W G T TS Q V K N V I W G T T S L I Q S V I W G T T S Q I Q S V I W G T T S L VQ S V I W G T T S Q V Q S V I W G T T S L I K S V I W G T T S Q I K S VI W G T T S L V K S V I W G T T S Q V K S V I W G T T S L I Q N I I W GT T S Q I Q N I I W G T T S L V Q N I I W G T T S Q V Q N I I W G T T SL I K N I I W G T T S Q I K N I I W G T T S L V K N I I W G T T S Q V KN I I W G T T S L I Q S I I W G T T S Q I Q S I I W G T T S L V Q S I IW G T T S Q V Q S I I W G T T S L I K S I I W G T T S Q I K S I I W G TT S L V K S I I W G T T S Q V K S I I W G T T S L I Q N V S W G T T S QI Q N V S W G T T S L V Q N V S W G T T S Q V Q N V S W G T T S L I K NV S W G T T S Q I K N V S W G T T S L V K N V S W G T T S Q V K N V S WG T T S L I Q S V S W G T T S Q I Q S V S W G T T S L V Q S V S W G T TS Q V Q S V S W G T T S L I K S V S W G T T S Q I K S V S W G T T S L VK S V S W G T T S Q V K S V S W G T T S L I Q N I S W G T T S Q I Q N IS W G T T S L V Q N I S W G T T S Q V Q N I S W G T T S L I K N I S W GT T S Q I K N I S W G T T S L V K N I S W G T T S Q V K N I S W G T T SL I Q S I S W G T T S Q I Q S I S W G T T S L V Q S I S W G T T S Q V QS I S W G T T S L I K S I S W G T T S Q I K S I S W G T T S L V K S I SW G T T S Q V K S I S W G T T S L I Q N V I Y D S S S Q I Q N V I Y D SS S L V Q N V I Y D S S S Q V Q N V I Y D S S S L I K N V I Y D S S S QI K N V I Y D S S S L V K N V I Y D S S S Q V K N V I Y D S S S L I Q SV I Y D S S S Q I Q S V I Y D S S S L V Q S V I Y D S S S Q V Q S V I YD S S S L I K S V I Y D S S S Q I K S V I Y D S S S L V K S V I Y D S SS Q V K S V I Y D S S S L I Q N I I Y D S S S Q I Q N I I Y D S S S L VQ N I I Y D S S S Q V Q N I I Y D S S S L I K N I I Y D S S S Q I K N II Y D S S S L V K N I I Y D S S S Q V K N I I Y D S S S L I Q S I I Y DS S S Q I Q S I I Y D S S S L V Q S I I Y D S S S Q V Q S I I Y D S S SL I K S I I Y D S S S Q I K S I I Y D S S S L V K S I I Y D S S S Q V KS I I Y D S S S L I Q N V S Y D S S S Q I Q N V S Y D S S S L V Q N V SY D S S S Q V Q N V S Y D S S S L I K N V S Y D S S S Q I K N V S Y D SS S L V K N V S Y D S S S Q V K N V S Y D S S S L I Q S V S Y D S S S QI Q S V S Y D S S S L V Q S V S Y D S S S Q V Q S V S Y D S S S L I K SV S Y D S S S Q I K S V S Y D S S S L V K S V S Y D S S S Q V K S V S YD S S S L I Q N I S Y D S S S Q I Q N I S Y D S S S L V Q N I S Y D S SS Q V Q N I S Y D S S S L I K N I S Y D S S S Q I K N I S Y D S S S L VK N I S Y D S S S Q V K N I S Y D S S S L I Q S I S Y D S S S Q I Q S IS Y D S S S L V Q S I S Y D S S S Q V Q S I S Y D S S S L I K S I S Y DS S S Q I K S I S Y D S S S L V K S I S Y D S S S Q V K S I S Y D S S SL I Q N V I W D S S S Q I Q N V I W D S S S L V Q N V I W D S S S Q V QN V I W D S S S L I K N V I W D S S S Q I K N V I W D S S S L V K N V IW D S S S Q V K N V I W D S S S L I Q S V I W D S S S Q I Q S V I W D SS S L V Q S V I W D S S S Q V Q S V I W D S S S L I K S V I W D S S S QI K S V I W D S S S L V K S V I W D S S S Q V K S V I W D S S S L I Q NI I W D S S S Q I Q N I I W D S S S L V Q N I I W D S S S Q V Q N I I WD S S S L I K N I I W D S S S Q I K N I I W D S S S L V K N I I W D S SS Q V K N I I W D S S S L I Q S I I W D S S S Q I Q S I I W D S S S L VQ S I I W D S S S Q V Q S I I W D S S S L I K S I I W D S S S Q I K S II W D S S S L V K S I I W D S S S Q V K S I I W D S S S L I Q N V S W DS S S Q I Q N V S W D S S S L V Q N V S W D S S S Q V Q N V S W D S S SL I K N V S W D S S S Q I K N V S W D S S S L V K N V S W D S S S Q V KN V S W D S S S L I Q S V S W D S S S Q I Q S V S W D S S S L V Q S V SW D S S S Q V Q S V S W D S S S L I K S V S W D S S S Q I K S V S W D SS S L V K S V S W D S S S Q V K S V S W D S S S L I Q N I S W D S S S QI Q N I S W D S S S L V Q N I S W D S S S Q V Q N I S W D S S S L I K NI S W D S S S Q I K N I S W D S S S L V K N I S W D S S S Q V K N I S WD S S S L I Q S I S W D S S S Q I Q S I S W D S S S L V Q S I S W D S SS Q V Q S I S W D S S S L I K S I S W D S S S Q I K S I S W D S S S L VK S I S W D S S S Q V K S I S W D S S S L I Q N V I Y G S S S Q I Q N VI Y G S S S L V Q N V I Y G S S S Q V Q N V I Y G S S S L I K N V I Y GS S S Q I K N V I Y G S S S L V K N V I Y G S S S Q V K N V I Y G S S SL I Q S V I Y G S S S Q I Q S V I Y G S S S L V Q S V I Y G S S S Q V QS V I Y G S S S L I K S V I Y G S S S Q I K S V I Y G S S S L V K S V IY G S S S Q V K S V I Y G S S S L I Q N I I Y G S S S Q I Q N I I Y G SS S L V Q N I I Y G S S S Q V Q N I I Y G S S S L I K N I I Y G S S S QI K N I I Y G S S S L V K N I I Y G S S S Q V K N I I Y G S S S L I Q SI I Y G S S S Q I Q S I I Y G S S S L V Q S I I Y G S S S Q V Q S I I YG S S S L I K S I I Y G S S S Q I K S I I Y G S S S L V K S I I Y G S SS Q V K S I I Y G S S S L I Q N V S Y G S S S Q I Q N V S Y G S S S L VQ N V S Y G S S S Q V Q N V S Y G S S S L I K N V S Y G S S S Q I K N VS Y G S S S L V K N V S Y G S S S Q V K N V S Y G S S S L I Q S V S Y GS S S Q I Q S V S Y G S S S L V Q S V S Y G S S S Q V Q S V S Y G S S SL I K S V S Y G S S S Q I K S V S Y G S S S L V K S V S Y G S S S Q V KS V S Y G S S S L I Q N I S Y G S S S Q I Q N I S Y G S S S L V Q N I SY G S S S Q V Q N I S Y G S S S L I K N I S Y G S S S Q I K N I S Y G SS S L V K N I S Y G S S S Q V K N I S Y G S S S L I Q S I S Y G S S S QI Q S I S Y G S S S L V Q S I S Y G S S S Q V Q S I S Y G S S S L I K SI S Y G S S S Q I K S I S Y G S S S L V K S I S Y G S S S Q V K S I S YG S S S L I Q N V I W G S S S Q I Q N V I W G S S S L V Q N V I W G S SS Q V Q N V I W G S S S L I K N V I W G S S S Q I K N V I W G S S S L VK N V I W G S S S Q V K N V I W G S S S L I Q S V I W G S S S Q I Q S VI W G S S S L V Q S V I W G S S S Q V Q S V I W G S S S L I K S V I W GS S S Q I K S V I W G S S S L V K S V I W G S S S Q V K S V I W G S S SL I Q N I I W G S S S Q I Q N I I W G S S S L V Q N I I W G S S S Q V QN I I W G S S S L I K N I I W G S S S Q I K N I I W G S S S L V K N I IW G S S S Q V K N I I W G S S S L I Q S I I W G S S S Q I Q S I I W G SS S L V Q S I I W G S S S Q V Q S I I W G S S S L I K S I I W G S S S QI K S I I W G S S S L V K S I I W G S S S Q V K S I I W G S S S L I Q NV S W G S S S Q I Q N V S W G S S S L V Q N V S W G S S S Q V Q N V S WG S S S L I K N V S W G S S S Q I K N V S W G S S S L V K N V S W G S SS Q V K N V S W G S S S L I Q S V S W G S S S Q I Q S V S W G S S S L VQ S V S W G S S S Q V Q S V S W G S S S L I K S V S W G S S S Q I K S VS W G S S S L V K S V S W G S S S Q V K S V S W G S S S L I Q N I S W GS S S Q I Q N I S W G S S S L V Q N I S W G S S S Q V Q N I S W G S S SL I K N I S W G S S S Q I K N I S W G S S S L V K N I S W G S S S Q V KN I S W G S S S L I Q S I S W G S S S Q I Q S I S W G S S S L V Q S I SW G S S S Q V Q S I S W G S S S L I K S I S W G S S S Q I K S I S W G SS S L V K S I S W G S S S Q V K S I S W G S S S L I Q N V I Y D T S S QI Q N V I Y D T S S L V Q N V I Y D T S S Q V Q N V I Y D T S S L I K NV I Y D T S S Q I K N V I Y D T S S L V K N V I Y D T S S Q V K N V I YD T S S L I Q S V I Y D T S S Q I Q S V I Y D T S S L V Q S V I Y D T SS Q V Q S V I Y D T S S L I K S V I Y D T S S Q I K S V I Y D T S S L VK S V I Y D T S S Q V K S V I Y D T S S L I Q N I I Y D T S S Q I Q N II Y D T S S L V Q N I I Y D T S S Q V Q N I I Y D T S S L I K N I I Y DT S S Q I K N I I Y D T S S L V K N I I Y D T S S Q V K N I I Y D T S SL I Q S I I Y D T S S Q I Q S I I Y D T S S L V Q S I I Y D T S S Q V QS I I Y D T S S L I K S I I Y D T S S Q I K S I I Y D T S S L V K S I IY D T S S Q V K S I I Y D T S S L I Q N V S Y D T S S Q I Q N V S Y D TS S L V Q N V S Y D T S S Q V Q N V S Y D T S S L I K N V S Y D T S S QI K N V S Y D T S S L V K N V S Y D T S S Q V K N V S Y D T S S L I Q SV S Y D T S S Q I Q S V S Y D T S S L V Q S V S Y D T S S Q V Q S V S YD T S S L I K S V S Y D T S S Q I K S V S Y D T S S L V K S V S Y D T SS Q V K S V S Y D T S S L I Q N I S Y D T S S Q I Q N I S Y D T S S L VQ N I S Y D T S S Q V Q N I S Y D T S S L I K N I S Y D T S S Q I K N IS Y D T S S L V K N I S Y D T S S Q V K N I S Y D T S S L I Q S I S Y DT S S Q I Q S I S Y D T S S L V Q S I S Y D T S S Q V Q S I S Y D T S SL I K S I S Y D T S S Q I K S I S Y D T S S L V K S I S Y D T S S Q V KS I S Y D T S S L I Q N V I W D T S S Q I Q N V I W D T S S L V Q N V IW D T S S Q V Q N V I W D T S S L I K N V I W D T S S Q I K N V I W D TS S L V K N V I W D T S S Q V K N V I W D T S S L I Q S V I W D T S S QI Q S V I W D T S S L V Q S V I W D T S S Q V Q S V I W D T S S L I K SV I W D T S S Q I K S V I W D T S S L V K S V I W D T S S Q V K S V I WD T S S L I Q N I I W D T S S Q I Q N I I W D T S S L V Q N I I W D T SS Q V Q N I I W D T S S L I K N I I W D T S S Q I K N I I W D T S S L VK N I I W D T S S Q V K N I I W D T S S L I Q S I I W D T S S Q I Q S II W D T S S L V Q S I I W D T S S Q V Q S I I W D T S S L I K S I I W DT S S Q I K S I I W D T S S L V K S I I W D T S S Q V K S I I W D T S SL I Q N V S W D T S S Q I Q N V S W D T S S L V Q N V S W D T S S Q V QN V S W D T S S L I K N V S W D T S S Q I K N V S W D T S S L V K N V SW D T S S Q V K N V S W D T S S L I Q S V S W D T S S Q I Q S V S W D TS S L V Q S V S W D T S S Q V Q S V S W D T S S L I K S V S W D T S S QI K S V S W D T S S L V K S V S W D T S S Q V K S V S W D T S S L I Q NI S W D T S S Q I Q N I S W D T S S L V Q N I S W D T S S Q V Q N I S WD T S S L I K N I S W D T S S Q I K N I S W D T S S L V K N I S W D T SS Q V K N I S W D T S S L I Q S I S W D T S S Q I Q S I S W D T S S L VQ S I S W D T S S Q V Q S I S W D T S S L I K S I S W D T S S Q I K S IS W D T S S L V K S I S W D T S S Q V K S I S W D T S S L I Q N V I Y GT S S Q I Q N V I Y G T S S L V Q N V I Y G T S S Q V Q N V I Y G T S SL I K N V I Y G T S S Q I K N V I Y G T S S L V K N V I Y G T S S Q V KN V I Y G T S S L I Q S V I Y G T S S Q I Q S V I Y G T S S L V Q S V IY G T S S Q V Q S V I Y G T S S L I K S V I Y G T S S Q I K S V I Y G TS S L V K S V I Y G T S S Q V K S V I Y G T S S L I Q N I I Y G T S S QI Q N I I Y G T S S L V Q N I I Y G T S S Q V Q N I I Y G T S S L I K NI I Y G T S S Q I K N I I Y G T S S L V K N I I Y G T S S Q V K N I I YG T S S L I Q S I I Y G T S S Q I Q S I I Y G T S S L V Q S I I Y G T SS Q V Q S I I Y G T S S L I K S I I Y G T S S Q I K S I I Y G T S S L VK S I I Y G T S S Q V K S I I Y G T S S L I Q N V S Y G T S S Q I Q N VS Y G T S S L V Q N V S Y G T S S Q V Q N V S Y G T S S L I K N V S Y GT S S Q I K N V S Y G T S S L V K N V S Y G T S S Q V K N V S Y G T S SL I Q S V S Y G T S S Q I Q S V S Y G T S S L V Q S V S Y G T S S Q V QS V S Y G T S S L I K S V S Y G T S S Q I K S V S Y G T S S L V K S V SY G T S S Q V K S V S Y G T S S L I Q N I S Y G T S S Q I Q N I S Y G TS S L V Q N I S Y G T S S Q V Q N I S Y G T S S L I K N I S Y G T S S QI K N I S Y G T S S L V K N I S Y G T S S Q V K N I S Y G T S S L I Q SI S Y G T S S Q I Q S I S Y G T S S L V Q S I S Y G T S S Q V Q S I S YG T S S L I K S I S Y G T S S Q I K S I S Y G T S S L V K S I S Y G T SS Q V K S I S Y G T S S L I Q N V I W G T S S Q I Q N V I W G T S S L VQ N V I W G T S S Q V Q N V I W G T S S L I K N V I W G T S S Q I K N VI W G T S S L V K N V I W G T S S Q V K N V I W G T S S L I Q S V I W GT S S Q I Q S V I W G T S S L V Q S V I W G T S S Q V Q S V I W G T S SL I K S V I W G T S S Q I K S V I W G T S S L V K S V I W G T S S Q V KS V I W G T S S L I Q N I I W G T S S Q I Q N I I W G T S S L V Q N I IW G T S S Q V Q N I I W G T S S L I K N I I W G T S S Q I K N I I W G TS S L V K N I I W G T S S Q V K N I I W G T S S L I Q S I I W G T S S QI Q S I I W G T S S L V Q S I I W G T S S Q V Q S I I W G T S S L I K SI I W G T S S Q I K S I I W G T S S L V K S I I W G T S S Q V K S I I WG T S S L I Q N V S W G T S S Q I Q N V S W G T S S L V Q N V S W G T SS Q V Q N V S W G T S S L I K N V S W G T S S Q I K N V S W G T S S L VK N V S W G T S S Q V K N V S W G T S S L I Q S V S W G T S S Q I Q S VS W G T S S L V Q S V S W G T S S Q V Q S V S W G T S S L I K S V S W GT S S Q I K S V S W G T S S L V K S V S W G T S S Q V K S V S W G T S SL I Q N I S W G T S S Q I Q N I S W G T S S L V Q N I S W G T S S Q V QN I S W G T S S L I K N I S W G T S S Q I K N I S W G T S S L V K N I SW G T S S Q V K N I S W G T S S L I Q S I S W G T S S Q I Q S I S W G TS S L V Q S I S W G T S S Q V Q S I S W G T S S L I K S I S W G T S S QI K S I S W G T S S L V K S I S W G T S S Q V K S I S W G T S S L I Q NV I Y D S T Y Q I Q N V I Y D S T Y L V Q N V I Y D S T Y Q V Q N V I YD S T Y L I K N V I Y D S T Y Q I K N V I Y D S T Y L V K N V I Y D S TY Q V K N V I Y D S T Y L I Q S V I Y D S T Y Q I Q S V I Y D S T Y L VQ S V I Y D S T Y Q V Q S V I Y D S T Y L I K S V I Y D S T Y Q I K S VI Y D S T Y L V K S V I Y D S T Y Q V K S V I Y D S T Y L I Q N I I Y DS T Y Q I Q N I I Y D S T Y L V Q N I I Y D S T Y Q V Q N I I Y D S T YL I K N I I Y D S T Y Q I K N I I Y D S T Y L V K N I I Y D S T Y Q V KN I I Y D S T Y L I Q S I I Y D S T Y Q I Q S I I Y D S T Y L V Q S I IY D S T Y Q V Q S I I Y D S T Y L I K S I I Y D S T Y Q I K S I I Y D ST Y L V K S I I Y D S T Y Q V K S I I Y D S T Y L I Q N V S Y D S T Y QI Q N V S Y D S T Y L V Q N V S Y D S T Y Q V Q N V S Y D S T Y L I K NV S Y D S T Y Q I K N V S Y D S T Y L V K N V S Y D S T Y Q V K N V S YD S T Y L I Q S V S Y D S T Y Q I Q S V S Y D S T Y L V Q S V S Y D S TY Q V Q S V S Y D S T Y L I K S V S Y D S T Y Q I K S V S Y D S T Y L VK S V S Y D S T Y Q V K S V S Y D S T Y L I Q N I S Y D S T Y Q I Q N IS Y D S T Y L V Q N I S Y D S T Y Q V Q N I S Y D S T Y L I K N I S Y DS T Y Q I K N I S Y D S T Y L V K N I S Y D S T Y Q V K N I S Y D S T YL I Q S I S Y D S T Y Q I Q S I S Y D S T Y L V Q S I S Y D S T Y Q V QS I S Y D S T Y L I K S I S Y D S T Y Q I K S I S Y D S T Y L V K S I SY D S T Y Q V K S I S Y D S T Y L I Q N V I W D S T Y Q I Q N V I W D ST Y L V Q N V I W D S T Y Q V Q N V I W D S T Y L I K N V I W D S T Y QI K N V I W D S T Y L V K N V I W D S T Y Q V K N V I W D S T Y L I Q SV I W D S T Y Q I Q S V I W D S T Y L V Q S V I W D S T Y Q V Q S V I WD S T Y L I K S V I W D S T Y Q I K S V I W D S T Y L V K S V I W D S TY Q V K S V I W D S T Y L I Q N I I W D S T Y Q I Q N I I W D S T Y L VQ N I I W D S T Y Q V Q N I I W D S T Y L I K N I I W D S T Y Q I K N II W D S T Y L V K N I I W D S T Y Q V K N I I W D S T Y L I Q S I I W DS T Y Q I Q S I I W D S T Y L V Q S I I W D S T Y Q V Q S I I W D S T YL I K S I I W D S T Y Q I K S I I W D S T Y L V K S I I W D S T Y Q V KS I I W D S T Y L I Q N V S W D S T Y Q I Q N V S W D S T Y L V Q N V SW D S T Y Q V Q N V S W D S T Y L I K N V S W D S T Y Q I K N V S W D ST Y L V K N V S W D S T Y Q V K N V S W D S T Y L I Q S V S W D S T Y QI Q S V S W D S T Y L V Q S V S W D S T Y Q V Q S V S W D S T Y L I K SV S W D S T Y Q I K S V S W D S T Y L V K S V S W D S T Y Q V K S V S WD S T Y L I Q N I S W D S T Y Q I Q N I S W D S T Y L V Q N I S W D S TY Q V Q N I S W D S T Y L I K N I S W D S T Y Q I K N I S W D S T Y L VK N I S W D S T Y Q V K N I S W D S T Y L I Q S I S W D S T Y Q I Q S IS W D S T Y L V Q S I S W D S T Y Q V Q S I S W D S T Y L I K S I S W DS T Y Q I K S I S W D S T Y L V K S I S W D S T Y Q V K S I S W D S T YL I Q N V I Y G S T Y Q I Q N V I Y G S T Y L V Q N V I Y G S T Y Q V QN V I Y G S T Y L I K N V I Y G S T Y Q I K N V I Y G S T Y L V K N V IY G S T Y Q V K N V I Y G S T Y L I Q S V I Y G S T Y Q I Q S V I Y G ST Y L V Q S V I Y G S T Y Q V Q S V I Y G S T Y L I K S V I Y G S T Y QI K S V I Y G S T Y L V K S V I Y G S T Y Q V K S V I Y G S T Y L I Q NI I Y G S T Y Q I Q N I I Y G S T Y L V Q N I I Y G S T Y Q V Q N I I YG S T Y L I K N I I Y G S T Y Q I K N I I Y G S T Y L V K N I I Y G S TY Q V K N I I Y G S T Y L I Q S I I Y G S T Y Q I Q S I I Y G S T Y L VQ S I I Y G S T Y Q V Q S I I Y G S T Y L I K S I I Y G S T Y Q I K S II Y G S T Y L V K S I I Y G S T Y Q V K S I I Y G S T Y L I Q N V S Y GS T Y Q I Q N V S Y G S T Y L V Q N V S Y G S T Y Q V Q N V S Y G S T YL I K N V S Y G S T Y Q I K N V S Y G S T Y L V K N V S Y G S T Y Q V KN V S Y G S T Y L I Q S V S Y G S T Y Q I Q S V S Y G S T Y L V Q S V SY G S T Y Q V Q S V S Y G S T Y L I K S V S Y G S T Y Q I K S V S Y G ST Y L V K S V S Y G S T Y Q V K S V S Y G S T Y L I Q N I S Y G S T Y QI Q N I S Y G S T Y L V Q N I S Y G S T Y Q V Q N I S Y G S T Y L I K NI S Y G S T Y Q I K N I S Y G S T Y L V K N I S Y G S T Y Q V K N I S YG S T Y L I Q S I S Y G S T Y Q I Q S I S Y G S T Y L V Q S I S Y G S TY Q V Q S I S Y G S T Y L I K S I S Y G S T Y Q I K S I S Y G S T Y L VK S I S Y G S T Y Q V K S I S Y G S T Y L I Q N V I W G S T Y Q I Q N VI W G S T Y L V Q N V I W G S T Y Q V Q N V I W G S T Y L I K N V I W GS T Y Q I K N V I W G S T Y L V K N V I W G S T Y Q V K N V I W G S T YL I Q S V I W G S T Y Q I Q S V I W G S T Y L V Q S V I W G S T Y Q V QS V I W G S T Y L I K S V I W G S T Y Q I K S V I W G S T Y L V K S V IW G S T Y Q V K S V I W G S T Y L I Q N I I W G S T Y Q I Q N I I W G ST Y L V Q N I I W G S T Y Q V Q N I I W G S T Y L I K N I I W G S T Y QI K N I I W G S T Y L V K N I I W G S T Y Q V K N I I W G S T Y L I Q SI I W G S T Y Q I Q S I I W G S T Y L V Q S I I W G S T Y Q V Q S I I WG S T Y L I K S I I W G S T Y Q I K S I I W G S T Y L V K S I I W G S TY Q V K S I I W G S T Y L I Q N V S W G S T Y Q I Q N V S W G S T Y L VQ N V S W G S T Y Q V Q N V S W G S T Y L I K N V S W G S T Y Q I K N VS W G S T Y L V K N V S W G S T Y Q V K N V S W G S T Y L I Q S V S W GS T Y Q I Q S V S W G S T Y L V Q S V S W G S T Y Q V Q S V S W G S T YL I K S V S W G S T Y Q I K S V S W G S T Y L V K S V S W G S T Y Q V KS V S W G S T Y L I Q N I S W G S T Y Q I Q N I S W G S T Y L V Q N I SW G S T Y Q V Q N I S W G S T Y L I K N I S W G S T Y Q I K N I S W G ST Y L V K N I S W G S T Y Q V K N I S W G S T Y L I Q S I S W G S T Y QI Q S I S W G S T Y L V Q S I S W G S T Y Q V Q S I S W G S T Y L I K SI S W G S T Y Q I K S I S W G S T Y L V K S I S W G S T Y Q V K S I S WG S T Y L I Q N V I Y D T T Y Q I Q N V I Y D T T Y L V Q N V I Y D T TY Q V Q N V I Y D T T Y L I K N V I Y D T T Y Q I K N V I Y D T T Y L VK N V I Y D T T Y Q V K N V I Y D T T Y L I Q S V I Y D T T Y Q I Q S VI Y D T T Y L V Q S V I Y D T T Y Q V Q S V I Y D T T Y L I K S V I Y DT T Y Q I K S V I Y D T T Y L V K S V I Y D T T Y Q V K S V I Y D T T YL I Q N I I Y D T T Y Q I Q N I I Y D T T Y L V Q N I I Y D T T Y Q V QN I I Y D T T Y L I K N I I Y D T T Y Q I K N I I Y D T T Y L V K N I IY D T T Y Q V K N I I Y D T T Y L I Q S I I Y D T T Y Q I Q S I I Y D TT Y L V Q S I I Y D T T Y Q V Q S I I Y D T T Y L I K S I I Y D T T Y QI K S I I Y D T T Y L V K S I I Y D T T Y Q V K S I I Y D T T Y L I Q NV S Y D T T Y Q I Q N V S Y D T T Y L V Q N V S Y D T T Y Q V Q N V S YD T T Y L I K N V S Y D T T Y Q I K N V S Y D T T Y L V K N V S Y D T TY Q V K N V S Y D T T Y L I Q S V S Y D T T Y Q I Q S V S Y D T T Y L VQ S V S Y D T T Y Q V Q S V S Y D T T Y L I K S V S Y D T T Y Q I K S VS Y D T T Y L V K S V S Y D T T Y Q V K S V S Y D T T Y L I Q N I S Y DT T Y Q I Q N I S Y D T T Y L V Q N I S Y D T T Y Q V Q N I S Y D T T YL I K N I S Y D T T Y Q I K N I S Y D T T Y L V K N I S Y D T T Y Q V KN I S Y D T T Y L I Q S I S Y D T T Y Q I Q S I S Y D T T Y L V Q S I SY D T T Y Q V Q S I S Y D T T Y L I K S I S Y D T T Y Q I K S I S Y D TT Y L V K S I S Y D T T Y Q V K S I S Y D T T Y L I Q N V I W D T T Y QI Q N V I W D T T Y L V Q N V I W D T T Y Q V Q N V I W D T T Y L I K NV I W D T T Y Q I K N V I W D T T Y L V K N V I W D T T Y Q V K N V I WD T T Y L I Q S V I W D T T Y Q I Q S V I W D T T Y L V Q S V I W D T TY Q V Q S V I W D T T Y L I K S V I W D T T Y Q I K S V I W D T T Y L VK S V I W D T T Y Q V K S V I W D T T Y L I Q N I I W D T T Y Q I Q N II W D T T Y L V Q N I I W D T T Y Q V Q N I I W D T T Y L I K N I I W DT T Y Q I K N I I W D T T Y L V K N I I W D T T Y Q V K N I I W D T T YL I Q S I I W D T T Y Q I Q S I I W D T T Y L V Q S I I W D T T Y Q V QS I I W D T T Y L I K S I I W D T T Y Q I K S I I W D T T Y L V K S I IW D T T Y Q V K S I I W D T T Y L I Q N V S W D T T Y Q I Q N V S W D TT Y L V Q N V S W D T T Y Q V Q N V S W D T T Y L I K N V S W D T T Y QI K N V S W D T T Y L V K N V S W D T T Y Q V K N V S W D T T Y L I Q SV S W D T T Y Q I Q S V S W D T T Y L V Q S V S W D T T Y Q V Q S V S WD T T Y L I K S V S W D T T Y Q I K S V S W D T T Y L V K S V S W D T TY Q V K S V S W D T T Y L I Q N I S W D T T Y Q I Q N I S W D T T Y L VQ N I S W D T T Y Q V Q N I S W D T T Y L I K N I S W D T T Y Q I K N IS W D T T Y L V K N I S W D T T Y Q V K N I S W D T T Y L I Q S I S W DT T Y Q I Q S I S W D T T Y L V Q S I S W D T T Y Q V Q S I S W D T T YL I K S I S W D T T Y Q I K S I S W D T T Y L V K S I S W D T T Y Q V KS I S W D T T Y L I Q N V I Y G T T Y Q I Q N V I Y G T T Y L V Q N V IY G T T Y Q V Q N V I Y G T T Y L I K N V I Y G T T Y Q I K N V I Y G TT Y L V K N V I Y G T T Y Q V K N V I Y G T T Y L I Q S V I Y G T T Y QI Q S V I Y G T T Y L V Q S V I Y G T T Y Q V Q S V I Y G T T Y L I K SV I Y G T T Y Q I K S V I Y G T T Y L V K S V I Y G T T Y Q V K S V I YG T T Y L I Q N I I Y G T T Y Q I Q N I I Y G T T Y L V Q N I I Y G T TY Q V Q N I I Y G T T Y L I K N I I Y G T T Y Q I K N I I Y G T T Y L VK N I I Y G T T Y Q V K N I I Y G T T Y L I Q S I I Y G T T Y Q I Q S II Y G T T Y L V Q S I I Y G T T Y Q V Q S I I Y G T T Y L I K S I I Y GT T Y Q I K S I I Y G T T Y L V K S I I Y G T T Y Q V K S I I Y G T T YL I Q N V S Y G T T Y Q I Q N V S Y G T T Y L V Q N V S Y G T T Y Q V QN V S Y G T T Y L I K N V S Y G T T Y Q I K N V S Y G T T Y L V K N V SY G T T Y Q V K N V S Y G T T Y L I Q S V S Y G T T Y Q I Q S V S Y G TT Y L V Q S V S Y G T T Y Q V Q S V S Y G T T Y L I K S V S Y G T T Y QI K S V S Y G T T Y L V K S V S Y G T T Y Q V K S V S Y G T T Y L I Q NI S Y G T T Y Q I Q N I S Y G T T Y L V Q N I S Y G T T Y Q V Q N I S YG T T Y L I K N I S Y G T T Y Q I K N I S Y G T T Y L V K N I S Y G T TY Q V K N I S Y G T T Y L I Q S I S Y G T T Y Q I Q S I S Y G T T Y L VQ S I S Y G T T Y Q V Q S I S Y G T T Y L I K S I S Y G T T Y Q I K S IS Y G T T Y L V K S I S Y G T T Y Q V K S I S Y G T T Y L I Q N V I W GT T Y Q I Q N V I W G T T Y L V Q N V I W G T T Y Q V Q N V I W G T T YL I K N V I W G T T Y Q I K N V I W G T T Y L V K N V I W G T T Y Q V KN V I W G T T Y L I Q S V I W G T T Y Q I Q S V I W G T T Y L V Q S V IW G T T Y Q V Q S V I W G T T Y L I K S V I W G T T Y Q I K S V I W G TT Y L V K S V I W G T T Y Q V K S V I W G T T Y L I Q N I I W G T T Y QI Q N I I W G T T Y L V Q N I I W G T T Y Q V Q N I I W G T T Y L I K NI I W G T T Y Q I K N I I W G T T Y L V K N I I W G T T Y Q V K N I I WG T T Y L I Q S I I W G T T Y Q I Q S I I W G T T Y L V Q S I I W G T TY Q V Q S I I W G T T Y L I K S I I W G T T Y Q I K S I I W G T T Y L VK S I I W G T T Y Q V K S I I W G T T Y L I Q N V S W G T T Y Q I Q N VS W G T T Y L V Q N V S W G T T Y Q V Q N V S W G T T Y L I K N V S W GT T Y Q I K N V S W G T T Y L V K N V S W G T T Y Q V K N V S W G T T YL I Q S V S W G T T Y Q I Q S V S W G T T Y L V Q S V S W G T T Y Q V QS V S W G T T Y L I K S V S W G T T Y Q I K S V S W G T T Y L V K S V SW G T T Y Q V K S V S W G T T Y L I Q N I S W G T T Y Q I Q N I S W G TT Y L V Q N I S W G T T Y Q V Q N I S W G T T Y L I K N I S W G T T Y QI K N I S W G T T Y L V K N I S W G T T Y Q V K N I S W G T T Y L I Q SI S W G T T Y Q I Q S I S W G T T Y L V Q S I S W G T T Y Q V Q S I S WG T T Y L I K S I S W G T T Y Q I K S I S W G T T Y L V K S I S W G T TY Q V K S I S W G T T Y L I Q N V I Y D S S Y Q I Q N V I Y D S S Y L VQ N V I Y D S S Y Q V Q N V I Y D S S Y L I K N V I Y D S S Y Q I K N VI Y D S S Y L V K N V I Y D S S Y Q V K N V I Y D S S Y L I Q S V I Y DS S Y Q I Q S V I Y D S S Y L V Q S V I Y D S S Y Q V Q S V I Y D S S YL I K S V I Y D S S Y Q I K S V I Y D S S Y L V K S V I Y D S S Y Q V KS V I Y D S S Y L I Q N I I Y D S S Y Q I Q N I I Y D S S Y L V Q N I IY D S S Y Q V Q N I I Y D S S Y L I K N I I Y D S S Y Q I K N I I Y D SS Y L V K N I I Y D S S Y Q V K N I I Y D S S Y L I Q S I I Y D S S Y QI Q S I I Y D S S Y L V Q S I I Y D S S Y Q V Q S I I Y D S S Y L I K SI I Y D S S Y Q I K S I I Y D S S Y L V K S I I Y D S S Y Q V K S I I YD S S Y L I Q N V S Y D S S Y Q I Q N V S Y D S S Y L V Q N V S Y D S SY Q V Q N V S Y D S S Y L I K N V S Y D S S Y Q I K N V S Y D S S Y L VK N V S Y D S S Y Q V K N V S Y D S S Y L I Q S V S Y D S S Y Q I Q S VS Y D S S Y L V Q S V S Y D S S Y Q V Q S V S Y D S S Y L I K S V S Y DS S Y Q I K S V S Y D S S Y L V K S V S Y D S S Y Q V K S V S Y D S S YL I Q N I S Y D S S Y Q I Q N I S Y D S S Y L V Q N I S Y D S S Y Q V QN I S Y D S S Y L I K N I S Y D S S Y Q I K N I S Y D S S Y L V K N I SY D S S Y Q V K N I S Y D S S Y L I Q S I S Y D S S Y Q I Q S I S Y D SS Y L V Q S I S Y D S S Y Q V Q S I S Y D S S Y L I K S I S Y D S S Y QI K S I S Y D S S Y L V K S I S Y D S S Y Q V K S I S Y D S S Y L I Q NV I W D S S Y Q I Q N V I W D S S Y L V Q N V I W D S S Y Q V Q N V I WD S S Y L I K N V I W D S S Y Q I K N V I W D S S Y L V K N V I W D S SY Q V K N V I W D S S Y L I Q S V I W D S S Y Q I Q S V I W D S S Y L VQ S V I W D S S Y Q V Q S V I W D S S Y L I K S V I W D S S Y Q I K S VI W D S S Y L V K S V I W D S S Y Q V K S V I W D S S Y L I Q N I I W DS S Y Q I Q N I I W D S S Y L V Q N I I W D S S Y Q V Q N I I W D S S YL I K N I I W D S S Y Q I K N I I W D S S Y L V K N I I W D S S Y Q V KN I I W D S S Y L I Q S I I W D S S Y Q I Q S I I W D S S Y L V Q S I IW D S S Y Q V Q S I I W D S S Y L I K S I I W D S S Y Q I K S I I W D SS Y L V K S I I W D S S Y Q V K S I I W D S S Y L I Q N V S W D S S Y QI Q N V S W D S S Y L V Q N V S W D S S Y Q V Q N V S W D S S Y L I K NV S W D S S Y Q I K N V S W D S S Y L V K N V S W D S S Y Q V K N V S WD S S Y L I Q S V S W D S S Y Q I Q S V S W D S S Y L V Q S V S W D S SY Q V Q S V S W D S S Y L I K S V S W D S S Y Q I K S V S W D S S Y L VK S V S W D S S Y Q V K S V S W D S S Y L I Q N I S W D S S Y Q I Q N IS W D S S Y L V Q N I S W D S S Y Q V Q N I S W D S S Y L I K N I S W DS S Y Q I K N I S W D S S Y L V K N I S W D S S Y Q V K N I S W D S S YL I Q S I S W D S S Y Q I Q S I S W D S S Y L V Q S I S W D S S Y Q V QS I S W D S S Y L I K S I S W D S S Y Q I K S I S W D S S Y L V K S I SW D S S Y Q V K S I S W D S S Y L I Q N V I Y G S S Y Q I Q N V I Y G SS Y L V Q N V I Y G S S Y Q V Q N V I Y G S S Y L I K N V I Y G S S Y QI K N V I Y G S S Y L V K N V I Y G S S Y Q V K N V I Y G S S Y L I Q SV I Y G S S Y Q I Q S V I Y G S S Y L V Q S V I Y G S S Y Q V Q S V I YG S S Y L I K S V I Y G S S Y Q I K S V I Y G S S Y L V K S V I Y G S SY Q V K S V I Y G S S Y L I Q N I I Y G S S Y Q I Q N I I Y G S S Y L VQ N I I Y G S S Y Q V Q N I I Y G S S Y L I K N I I Y G S S Y Q I K N II Y G S S Y L V K N I I Y G S S Y Q V K N I I Y G S S Y L I Q S I I Y GS S Y Q I Q S I I Y G S S Y L V Q S I I Y G S S Y Q V Q S I I Y G S S YL I K S I I Y G S S Y Q I K S I I Y G S S Y L V K S I I Y G S S Y Q V KS I I Y G S S Y L I Q N V S Y G S S Y Q I Q N V S Y G S S Y L V Q N V SY G S S Y Q V Q N V S Y G S S Y L I K N V S Y G S S Y Q I K N V S Y G SS Y L V K N V S Y G S S Y Q V K N V S Y G S S Y L I Q S V S Y G S S Y QI Q S V S Y G S S Y L V Q S V S Y G S S Y Q V Q S V S Y G S S Y L I K SV S Y G S S Y Q I K S V S Y G S S Y L V K S V S Y G S S Y Q V K S V S YG S S Y L I Q N I S Y G S S Y Q I Q N I S Y G S S Y L V Q N I S Y G S SY Q V Q N I S Y G S S Y L I K N I S Y G S S Y Q I K N I S Y G S S Y L VK N I S Y G S S Y Q V K N I S Y G S S Y L I Q S I S Y G S S Y Q I Q S IS Y G S S Y L V Q S I S Y G S S Y Q V Q S I S Y G S S Y L I K S I S Y GS S Y Q I K S I S Y G S S Y L V K S I S Y G S S Y Q V K S I S Y G S S YL I Q N V I W G S S Y Q I Q N V I W G S S Y L V Q N V I W G S S Y Q V QN V I W G S S Y L I K N V I W G S S Y Q I K N V I W G S S Y L V K N V IW G S S Y Q V K N V I W G S S Y L I Q S V I W G S S Y Q I Q S V I W G SS Y L V Q S V I W G S S Y Q V Q S V I W G S S Y L I K S V I W G S S Y QI K S V I W G S S Y L V K S V I W G S S Y Q V K S V I W G S S Y L I Q NI I W G S S Y Q I Q N I I W G S S Y L V Q N I I W G S S Y Q V Q N I I WG S S Y L I K N I I W G S S Y Q I K N I I W G S S Y L V K N I I W G S SY Q V K N I I W G S S Y L I Q S I I W G S S Y Q I Q S I I W G S S Y L VQ S I I W G S S Y Q V Q S I I W G S S Y L I K S I I W G S S Y Q I K S II W G S S Y L V K S I I W G S S Y Q V K S I I W G S S Y L I Q N V S W GS S Y Q I Q N V S W G S S Y L V Q N V S W G S S Y Q V Q N V S W G S S YL I K N V S W G S S Y Q I K N V S W G S S Y L V K N V S W G S S Y Q V KN V S W G S S Y L I Q S V S W G S S Y Q I Q S V S W G S S Y L V Q S V SW G S S Y Q V Q S V S W G S S Y L I K S V S W G S S Y Q I K S V S W G SS Y L V K S V S W G S S Y Q V K S V S W G S S Y L I Q N I S W G S S Y QI Q N I S W G S S Y L V Q N I S W G S S Y Q V Q N I S W G S S Y L I K NI S W G S S Y Q I K N I S W G S S Y L V K N I S W G S S Y Q V K N I S WG S S Y L I Q S I S W G S S Y Q I Q S I S W G S S Y L V Q S I S W G S SY Q V Q S I S W G S S Y L I K S I S W G S S Y Q I K S I S W G S S Y L VK S I S W G S S Y Q V K S I S W G S S Y L I Q N V I Y D T S Y Q I Q N VI Y D T S Y L V Q N V I Y D T S Y Q V Q N V I Y D T S Y L I K N V I Y DT S Y Q I K N V I Y D T S Y L V K N V I Y D T S Y Q V K N V I Y D T S YL I Q S V I Y D T S Y Q I Q S V I Y D T S Y L V Q S V I Y D T S Y Q V QS V I Y D T S Y L I K S V I Y D T S Y Q I K S V I Y D T S Y L V K S V IY D T S Y Q V K S V I Y D T S Y L I Q N I I Y D T S Y Q I Q N I I Y D TS Y L V Q N I I Y D T S Y Q V Q N I I Y D T S Y L I K N I I Y D T S Y QI K N I I Y D T S Y L V K N I I Y D T S Y Q V K N I I Y D T S Y L I Q SI I Y D T S Y Q I Q S I I Y D T S Y L V Q S I I Y D T S Y Q V Q S I I YD T S Y L I K S I I Y D T S Y Q I K S I I Y D T S Y L V K S I I Y D T SY Q V K S I I Y D T S Y L I Q N V S Y D T S Y Q I Q N V S Y D T S Y L VQ N V S Y D T S Y Q V Q N V S Y D T S Y L I K N V S Y D T S Y Q I K N VS Y D T S Y L V K N V S Y D T S Y Q V K N V S Y D T S Y L I Q S V S Y DT S Y Q I Q S V S Y D T S Y L V Q S V S Y D T S Y Q V Q S V S Y D T S YL I K S V S Y D T S Y Q I K S V S Y D T S Y L V K S V S Y D T S Y Q V KS V S Y D T S Y L I Q N I S Y D T S Y Q I Q N I S Y D T S Y L V Q N I SY D T S Y Q V Q N I S Y D T S Y L I K N I S Y D T S Y Q I K N I S Y D TS Y L V K N I S Y D T S Y Q V K N I S Y D T S Y L I Q S I S Y D T S Y QI Q S I S Y D T S Y L V Q S I S Y D T S Y Q V Q S I S Y D T S Y L I K SI S Y D T S Y Q I K S I S Y D T S Y L V K S I S Y D T S Y Q V K S I S YD T S Y L I Q N V I W D T S Y Q I Q N V I W D T S Y L V Q N V I W D T SY Q V Q N V I W D T S Y L I K N V I W D T S Y Q I K N V I W D T S Y L VK N V I W D T S Y Q V K N V I W D T S Y L I Q S V I W D T S Y Q I Q S VI W D T S Y L V Q S V I W D T S Y Q V Q S V I W D T S Y L I K S V I W DT S Y Q I K S V I W D T S Y L V K S V I W D T S Y Q V K S V I W D T S YL I Q N I I W D T S Y Q I Q N I I W D T S Y L V Q N I I W D T S Y Q V QN I I W D T S Y L I K N I I W D T S Y Q I K N I I W D T S Y L V K N I IW D T S Y Q V K N I I W D T S Y L I Q S I I W D T S Y Q I Q S I I W D TS Y L V Q S I I W D T S Y Q V Q S I I W D T S Y L I K S I I W D T S Y QI K S I I W D T S Y L V K S I I W D T S Y Q V K S I I W D T S Y L I Q NV S W D T S Y Q I Q N V S W D T S Y L V Q N V S W D T S Y Q V Q N V S WD T S Y L I K N V S W D T S Y Q I K N V S W D T S Y L V K N V S W D T SY Q V K N V S W D T S Y L I Q S V S W D T S Y Q I Q S V S W D T S Y L VQ S V S W D T S Y Q V Q S V S W D T S Y L I K S V S W D T S Y Q I K S VS W D T S Y L V K S V S W D T S Y Q V K S V S W D T S Y L I Q N I S W DT S Y Q I Q N I S W D T S Y L V Q N I S W D T S Y Q V Q N I S W D T S YL I K N I S W D T S Y Q I K N I S W D T S Y L V K N I S W D T S Y Q V KN I S W D T S Y L I Q S I S W D T S Y Q I Q S I S W D T S Y L V Q S I SW D T S Y Q V Q S I S W D T S Y L I K S I S W D T S Y Q I K S I S W D TS Y L V K S I S W D T S Y Q V K S I S W D T S Y L I Q N V I Y G T S Y QI Q N V I Y G T S Y L V Q N V I Y G T S Y Q V Q N V I Y G T S Y L I K NV I Y G T S Y Q I K N V I Y G T S Y L V K N V I Y G T S Y Q V K N V I YG T S Y L I Q S V I Y G T S Y Q I Q S V I Y G T S Y L V Q S V I Y G T SY Q V Q S V I Y G T S Y L I K S V I Y G T S Y Q I K S V I Y G T S Y L VK S V I Y G T S Y Q V K S V I Y G T S Y L I Q N I I Y G T S Y Q I Q N II Y G T S Y L V Q N I I Y G T S Y Q V Q N I I Y G T S Y L I K N I I Y GT S Y Q I K N I I Y G T S Y L V K N I I Y G T S Y Q V K N I I Y G T S YL I Q S I I Y G T S Y Q I Q S I I Y G T S Y L V Q S I I Y G T S Y Q V QS I I Y G T S Y L I K S I I Y G T S Y Q I K S I I Y G T S Y L V K S I IY G T S Y Q V K S I I Y G T S Y L I Q N V S Y G T S Y Q I Q N V S Y G TS Y L V Q N V S Y G T S Y Q V Q N V S Y G T S Y L I K N V S Y G T S Y QI K N V S Y G T S Y L V K N V S Y G T S Y Q V K N V S Y G T S Y L I Q SV S Y G T S Y Q I Q S V S Y G T S Y L V Q S V S Y G T S Y Q V Q S V S YG T S Y L I K S V S Y G T S Y Q I K S V S Y G T S Y L V K S V S Y G T SY Q V K S V S Y G T S Y L I Q N I S Y G T S Y Q I Q N I S Y G T S Y L VQ N I S Y G T S Y Q V Q N I S Y G T S Y L I K N I S Y G T S Y Q I K N IS Y G T S Y L V K N I S Y G T S Y Q V K N I S Y G T S Y L I Q S I S Y GT S Y Q I Q S I S Y G T S Y L V Q S I S Y G T S Y Q V Q S I S Y G T S YL I K S I S Y G T S Y Q I K S I S Y G T S Y L V K S I S Y G T S Y Q V KS I S Y G T S Y L I Q N V I W G T S Y Q I Q N V I W G T S Y L V Q N V IW G T S Y Q V Q N V I W G T S Y L I K N V I W G T S Y Q I K N V I W G TS Y L V K N V I W G T S Y Q V K N V I W G T S Y L I Q S V I W G T S Y QI Q S V I W G T S Y L V Q S V I W G T S Y Q V Q S V I W G T S Y L I K SV I W G T S Y Q I K S V I W G T S Y L V K S V I W G T S Y Q V K S V I WG T S Y L I Q N I I W G T S Y Q I Q N I I W G T S Y L V Q N I I W G T SY Q V Q N I I W G T S Y L I K N I I W G T S Y Q I K N I I W G T S Y L VK N I I W G T S Y Q V K N I I W G T S Y L I Q S I I W G T S Y Q I Q S II W G T S Y L V Q S I I W G T S Y Q V Q S I I W G T S Y L I K S I I W GT S Y Q I K S I I W G T S Y L V K S I I W G T S Y Q V K S I I W G T S YL I Q N V S W G T S Y Q I Q N V S W G T S Y L V Q N V S W G T S Y Q V QN V S W G T S Y L I K N V S W G T S Y Q I K N V S W G T S Y L V K N V SW G T S Y Q V K N V S W G T S Y L I Q S V S W G T S Y Q I Q S V S W G TS Y L V Q S V S W G T S Y Q V Q S V S W G T S Y L I K S V S W G T S Y QI K S V S W G T S Y L V K S V S W G T S Y Q V K S V S W G T S Y L I Q NI S W G T S Y Q I Q N I S W G T S Y L V Q N I S W G T S Y Q V Q N I S WG T S Y L I K N I S W G T S Y Q I K N I S W G T S Y L V K N I S W G T SY Q V K N I S W G T S Y L I Q S I S W G T S Y Q I Q S I S W G T S Y L VQ S I S W G T S Y Q V Q S I S W G T S Y L I K S I S W G T S Y Q I K S IS W G T S Y L V K S I S W G T S Y Q V K S I S W G T S Y

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 2. In certainembodiments, SEQ ID NO.: 2 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 7. Insome embodiments, SEQ ID NO: 2 comprises any one, any two, any three,any four, any five, any six, any seven, any eight, any nine, any ten,any eleven, or all eleven of the germline residues as indicated in Table7. In certain embodiments, SEQ ID NO.: 2 comprises any one of the uniquecombinations of germline and non-germline residues indicated by each rowof Table 7. In other embodiments, the targeted binding agent or antibodyis derived from a germline sequence with VH1-18, D7-27 and JH4 domains,wherein one or more residues has been mutated to yield the correspondinggermline residue at that position.

TABLE 8 Exemplary Mutations of 4B4 light Chain (SEQ ID NO: 4) toGermline at the Indicated Residue Number 51 88 92 95 97 I F N D S S F ND S I Y N D S S Y N D S I F S D S S F S D S I Y S D S S Y S D S I F N NS S F N N S I Y N N S S Y N N S I F S N S S F S N S I Y S N S S Y S N SI F N D V S F N D V I Y N D V S Y N D V I F S D V S F S D V I Y S D V SY S D V I F N N V S F N N V I Y N N V S Y N N V I F S N V S F S N V I YS N V S Y S N V

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 4. In certainembodiments, SEQ ID NO.: 4 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 8. Insome embodiments, SEQ ID NO: 4 comprises any one, any two, any three,any four, any five, or all five of the germline residues as indicated inTable 8. In certain embodiments, SEQ ID NO.: 4 comprises any one of theunique combinations of germline and non-germline residues indicated byeach row of Table 8. In other embodiments, the targeted binding agent orantibody is derived from a germline sequence with VL, 3p and JL2domains, wherein one or more residues has been mutated to yield thecorresponding germline residue at that position.

TABLE 9 Exemplary Mutations of 2H10 Heavy Chain (SEQ ID NO: 6) toGermline at the Indicated Residue Number 31 32 51 53 58 93 R H V F I M SH V F I M R Y V F I M S Y V F I M R H I F I M S H I F I M R Y I F I M SY I F I M R H V Y I M S H V Y I M R Y V Y I M S Y V Y I M R H I Y I M SH I Y I M R Y I Y I M S Y I Y I M R H V F K M S H V F K M R Y V F K M SY V F K M R H I F K M S H I F K M R Y I F K M S Y I F K M R H V Y K M SH V Y K M R Y V Y K M S Y V Y K M R H I Y K M S H I Y K M R Y I Y K M SY I Y K M R H V F I V S H V F I V R Y V F I V S Y V F I V R H I F I V SH I F I V R Y I F I V S Y I F I V R H V Y I V S H V Y I V R Y V Y I V SY V Y I V R H I Y I V S H I Y I V R Y I Y I V S Y I Y I V R H V F K V SH V F K V R Y V F K V S Y V F K V R H I F K V S H I F K V R Y I F K V SY I F K V R H V Y K V S H V Y K V R Y V Y K V S Y V Y K V R H I Y K V SH I Y K V R Y I Y K V S Y I Y K V

A further embodiment In some embodiments of the invention, the targetedbinding agent or antibody comprises a sequence comprising SEQ ID NO.: 6.In certain embodiments, SEQ ID NO.: 6 comprises any one of thecombinations of germline and non-germline residues indicated by each rowof Table 9. In some embodiments, SEQ ID NO: 6 comprises any one, anytwo, any three, any four, any five, any six or all six of the germlineresidues as indicated in Table 9. In certain embodiments, SEQ ID NO.: 6comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 9. In otherembodiments, the targeted binding agent or antibody is derived from agermline sequence with Vh3-33, D6-13 and JH4 domains, wherein one ormore residues has been mutated to yield the corresponding germlineresidue at that position. An example of a sequence, which has beenmutated is 2H10 VHOP where the M at position 93 has been mutated to a V.See Table 13.

TABLE 10 Exemplary Mutations of 2H10 light Chain (SEQ ID NO: 8) toGermline at the Indicated Residue Number 18 32 50 65 89 94 V V E S T L AV E S T L V A E S T L A A E S T L V V D S T L A V D S T L V A D S T L AA D S T L V V E N T L A V E N T L V A E N T L A A E N T L V V D N T L AV D N T L V A D N T L A A D N T L V V E S A L A V E S A L V A E S A L AA E S A L V V D S A L A V D S A L V A D S A L A A D S A L V V E N A L AV E N A L V A E N A L A A E N A L V V D N A L A V D N A L V A D N A L AA D N A L V V E S T T A V E S T T V A E S T T A A E S T T V V D S T T AV D S T T V A D S T T A A D S T T V V E N T T A V E N T T V A E N T T AA E N T T V V D N T T A V D N T T V A D N T T A A D N T T V V E S A T AV E S A T V A E S A T A A E S A T V V D S A T A V D S A T V A D S A T AA D S A T V V E N A T A V E N A T V A E N A T A A E N A T V V D N A T AV D N A T V A D N A T A A D N A T

A further embodiment In some embodiments of the invention, the targetedbinding agent or antibody comprises a sequence comprising SEQ ID NO.: 8.In certain embodiments, SEQ ID NO.: 8 comprises any one of thecombinations of germline and non-germline residues indicated by each rowof Table 10. In some embodiments, SEQ ID NO: 8 comprises any one, anytwo, any three, any four, any five, any six or all six of the germlineresidues as indicated in Table 10. In certain embodiments, SEQ ID NO.: 8comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 10. In otherembodiments, the targeted binding agent or antibody is derived from agermline sequence with VL, 3r and JL2 domains, wherein one or moreresidues has been mutated to yield the corresponding germline residue atthat position. In certain embodiments, SEQ ID NO.: 8 can comprisefurther modifications that include removing structural liabilities. Forexample, in addition to germlining, the C33 can be mutated to an S—seefor example 2H10 VLOP1 in Table 13. Thus, SEQ ID NO.: 8 can comprise anyone of the unique combinations of germline and non-germline residuesindicated by each row of Table 10 and further include the mutation ofC33 to a S. Examples of a sequence where the light chain has beenmutated to remove the structural liability at C33 and further mutatedback to the germline sequence is 2H10 VLOP2 as shown in Table 13 whereC33 has been mutated to an S and V at position 18 has been mutated to anA, and S at position 65 has been mutated to a N.

TABLE 11 Exemplary Mutations of 9G8 Heavy Chain (SEQ ID NO: 22) toGermline at the Indicated Residue Number 21 34 38 62 70 96 108 S S L S SF V T S L S S F V S Y L S S F V T Y L S S F V S S W S S F V T S W S S FV S Y W S S F V T Y W S S F V S S L N S F V T S L N S F V S Y L N S F VT Y L N S F V S S W N S F V T S W N S F V S Y W N S F V T Y W N S F V SS L S T F V T S L S T F V S Y L S T F V T Y L S T F V S S W S T F V T SW S T F V S Y W S T F V T Y W S T F V S S L N T F V T S L N T F V S Y LN T F V T Y L N T F V S S W N T F V T S W N T F V S Y W N T F V T Y W NT F V S S L S S Y V T S L S S Y V S Y L S S Y V T Y L S S Y V S S W S SY V T S W S S Y V S Y W S S Y V T Y W S S Y V S S L N S Y V T S L N S YV S Y L N S Y V T Y L N S Y V S S W N S Y V T S W N S Y V S Y W N S Y VT Y W N S Y V S S L S T Y V T S L S T Y V S Y L S T Y V T Y L S T Y V SS W S T Y V T S W S T Y V S Y W S T Y V T Y W S T Y V S S L N T Y V T SL N T Y V S Y L N T Y V T Y L N T Y V S S W N T Y V T S W N T Y V S Y WN T Y V T Y W N T Y V S S L S S F A T S L S S F A S Y L S S F A T Y L SS F A S S W S S F A T S W S S F A S Y W S S F A T Y W S S F A S S L N SF A T S L N S F A S Y L N S F A T Y L N S F A S S W N S F A T S W N S FA S Y W N S F A T Y W N S F A S S L S T F A T S L S T F A S Y L S T F AT Y L S T F A S S W S T F A T S W S T F A S Y W S T F A T Y W S T F A SS L N T F A T S L N T F A S Y L N T F A T Y L N T F A S S W N T F A T SW N T F A S Y W N T F A T Y W N T F A S S L S S Y A T S L S S Y A S Y LS S Y A T Y L S S Y A S S W S S Y A T S W S S Y A S Y W S S Y A T Y W SS Y A S S L N S Y A T S L N S Y A S Y L N S Y A T Y L N S Y A S S W N SY A T S W N S Y A S Y W N S Y A T Y W N S Y A S S L S T Y A T S L S T YA S Y L S T Y A T Y L S T Y A S S W S T Y A T S W S T Y A S Y W S T Y AT Y W S T Y A S S L N T Y A T S L N T Y A S Y L N T Y A T Y L N T Y A SS W N T Y A T S W N T Y A S Y W N T Y A T Y W N T Y A

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 22. In certainembodiments, SEQ ID NO.: 22 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 11. Insome embodiments, SEQ ID NO: 22 comprises any one, any two, any three,any four, any five, any six, any seven, or all seven of the germlineresidues as indicated in Table 11. In certain embodiments, SEQ ID NO.:22 comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 11. In otherembodiments, the targeted binding agent or antibody is derived from agermline sequence with VH4-39, D4-23 and JH3 domains, wherein one ormore residues has been mutated to yield the corresponding germlineresidue at that position. An example of a sequence where the heavy chainhas been mutated back to the germline sequence is 9G8 VHOP1 shown inTable 13 where L at position 38 has been mutated to a W. Another exampleof a sequence where the heavy chain has been mutated back to thegermline sequence is 9G8 VHOP2 shown in Table 13 where S at position 21has been mutated to a T, L at position 38 has been mutated to a W, S atposition 70 has been mutated to a T, and F at position 96 has beenmutated to a Y.

TABLE 12 Exemplary Mutations of 9G8 Light Chain (SEQ ID NO: 24) toGermline at the Indicated Residue Number 2 7 19 30 32 39 49 51 79 94 97S S R V V T E T V T I Y S R V V T E T V T I S P R V V T E T V T I Y P RV V T E T V T I S S S V V T E T V T I Y S S V V T E T V T I S P S V V TE T V T I Y P S V V T E T V T I S S R K V T E T V T I Y S R K V T E T VT I S P R K V T E T V T I Y P R K V T E T V T I S S S K V T E T V T I YS S K V T E T V T I S P S K V T E T V T I Y P S K V T E T V T I S S R VA T E T V T I Y S R V A T E T V T I S P R V A T E T V T I Y P R V A T ET V T I S S S V A T E T V T I Y S S V A T E T V T I S P S V A T E T V TI Y P S V A T E T V T I S S R K A T E T V T I Y S R K A T E T V T I S PR K A T E T V T I Y P R K A T E T V T I S S S K A T E T V T I Y S S K AT E T V T I S P S K A T E T V T I Y P S K A T E T V T I S S R V V P E TV T I Y S R V V P E T V T I S P R V V P E T V T I Y P R V V P E T V T IS S S V V P E T V T I Y S S V V P E T V T I S P S V V P E T V T I Y P SV V P E T V T I S S R K V P E T V T I Y S R K V P E T V T I S P R K V PE T V T I Y P R K V P E T V T I S S S K V P E T V T I Y S S K V P E T VT I S P S K V P E T V T I Y P S K V P E T V T I S S R V A P E T V T I YS R V A P E T V T I S P R V A P E T V T I Y P R V A P E T V T I S S S VA P E T V T I Y S S V A P E T V T I S P S V A P E T V T I Y P S V A P ET V T I S S R K A P E T V T I Y S R K A P E T V T I S P R K A P E T V TI Y P R K A P E T V T I S S S K A P E T V T I Y S S K A P E T V T I S PS K A P E T V T I Y P S K A P E T V T I S S R V V T Q T V T I Y S R V VT Q T V T I S P R V V T Q T V T I Y P R V V T Q T V T I S S S V V T Q TV T I Y S S V V T Q T V T I S P S V V T Q T V T I Y P S V V T Q T V T IS S R K V T Q T V T I Y S R K V T Q T V T I S P R K V T Q T V T I Y P RK V T Q T V T I S S S K V T Q T V T I Y S S K V T Q T V T I S P S K V TQ T V T I Y P S K V T Q T V T I S S R V A T Q T V T I Y S R V A T Q T VT I S P R V A T Q T V T I Y P R V A T Q T V T I S S S V A T Q T V T I YS S V A T Q T V T I S P S V A T Q T V T I Y P S V A T Q T V T I S S R KA T Q T V T I Y S R K A T Q T V T I S P R K A T Q T V T I Y P R K A T QT V T I S S S K A T Q T V T I Y S S K A T Q T V T I S P S K A T Q T V TI Y P S K A T Q T V T I S S R V V P Q T V T I Y S R V V P Q T V T I S PR V V P Q T V T I Y P R V V P Q T V T I S S S V V P Q T V T I Y S S V VP Q T V T I S P S V V P Q T V T I Y P S V V P Q T V T I S S R K V P Q TV T I Y S R K V P Q T V T I S P R K V P Q T V T I Y P R K V P Q T V T IS S S K V P Q T V T I Y S S K V P Q T V T I S P S K V P Q T V T I Y P SK V P Q T V T I S S R V A P Q T V T I Y S R V A P Q T V T I S P R V A PQ T V T I Y P R V A P Q T V T I S S S V A P Q T V T I Y S S V A P Q T VT I S P S V A P Q T V T I Y P S V A P Q T V T I S S R K A P Q T V T I YS R K A P Q T V T I S P R K A P Q T V T I Y P R K A P Q T V T I S S S KA P Q T V T I Y S S K A P Q T V T I S P S K A P Q T V T I Y P S K A P QT V T I S S R V V T E S V T I Y S R V V T E S V T I S P R V V T E S V TI Y P R V V T E S V T I S S S V V T E S V T I Y S S V V T E S V T I S PS V V T E S V T I Y P S V V T E S V T I S S R K V T E S V T I Y S R K VT E S V T I S P R K V T E S V T I Y P R K V T E S V T I S S S K V T E SV T I Y S S K V T E S V T I S P S K V T E S V T I Y P S K V T E S V T IS S R V A T E S V T I Y S R V A T E S V T I S P R V A T E S V T I Y P RV A T E S V T I S S S V A T E S V T I Y S S V A T E S V T I S P S V A TE S V T I Y P S V A T E S V T I S S R K A T E S V T I Y S R K A T E S VT I S P R K A T E S V T I Y P R K A T E S V T I S S S K A T E S V T I YS S K A T E S V T I S P S K A T E S V T I Y P S K A T E S V T I S S R VV P E S V T I Y S R V V P E S V T I S P R V V P E S V T I Y P R V V P ES V T I S S S V V P E S V T I Y S S V V P E S V T I S P S V V P E S V TI Y P S V V P E S V T I S S R K V P E S V T I Y S R K V P E S V T I S PR K V P E S V T I Y P R K V P E S V T I S S S K V P E S V T I Y S S K VP E S V T I S P S K V P E S V T I Y P S K V P E S V T I S S R V A P E SV T I Y S R V A P E S V T I S P R V A P E S V T I Y P R V A P E S V T IS S S V A P E S V T I Y S S V A P E S V T I S P S V A P E S V T I Y P SV A P E S V T I S S R K A P E S V T I Y S R K A P E S V T I S P R K A PE S V T I Y P R K A P E S V T I S S S K A P E S V T I Y S S K A P E S VT I S P S K A P E S V T I Y P S K A P E S V T I S S R V V T Q S V T I YS R V V T Q S V T I S P R V V T Q S V T I Y P R V V T Q S V T I S S S VV T Q S V T I Y S S V V T Q S V T I S P S V V T Q S V T I Y P S V V T QS V T I S S R K V T Q S V T I Y S R K V T Q S V T I S P R K V T Q S V TI Y P R K V T Q S V T I S S S K V T Q S V T I Y S S K V T Q S V T I S PS K V T Q S V T I Y P S K V T Q S V T I S S R V A T Q S V T I Y S R V AT Q S V T I S P R V A T Q S V T I Y P R V A T Q S V T I S S S V A T Q SV T I Y S S V A T Q S V T I S P S V A T Q S V T I Y P S V A T Q S V T IS S R K A T Q S V T I Y S R K A T Q S V T I S P R K A T Q S V T I Y P RK A T Q S V T I S S S K A T Q S V T I Y S S K A T Q S V T I S P S K A TQ S V T I Y P S K A T Q S V T I S S R V V P Q S V T I Y S R V V P Q S VT I S P R V V P Q S V T I Y P R V V P Q S V T I S S S V V P Q S V T I YS S V V P Q S V T I S P S V V P Q S V T I Y P S V V P Q S V T I S S R KV P Q S V T I Y S R K V P Q S V T I S P R K V P Q S V T I Y P R K V P QS V T I S S S K V P Q S V T I Y S S K V P Q S V T I S P S K V P Q S V TI Y P S K V P Q S V T I S S R V A P Q S V T I Y S R V A P Q S V T I S PR V A P Q S V T I Y P R V A P Q S V T I S S S V A P Q S V T I Y S S V AP Q S V T I S P S V A P Q S V T I Y P S V A P Q S V T I S S R K A P Q SV T I Y S R K A P Q S V T I S P R K A P Q S V T I Y P R K A P Q S V T IS S S K A P Q S V T I Y S S K A P Q S V T I S P S K A P Q S V T I Y P SK A P Q S V T I S S R V V T E T A T I Y S R V V T E T A T I S P R V V TE T A T I Y P R V V T E T A T I S S S V V T E T A T I Y S S V V T E T AT I S P S V V T E T A T I Y P S V V T E T A T I S S R K V T E T A T I YS R K V T E T A T I S P R K V T E T A T I Y P R K V T E T A T I S S S KV T E T A T I Y S S K V T E T A T I S P S K V T E T A T I Y P S K V T ET A T I S S R V A T E T A T I Y S R V A T E T A T I S P R V A T E T A TI Y P R V A T E T A T I S S S V A T E T A T I Y S S V A T E T A T I S PS V A T E T A T I Y P S V A T E T A T I S S R K A T E T A T I Y S R K AT E T A T I S P R K A T E T A T I Y P R K A T E T A T I S S S K A T E TA T I Y S S K A T E T A T I S P S K A T E T A T I Y P S K A T E T A T IS S R V V P E T A T I Y S R V V P E T A T I S P R V V P E T A T I Y P RV V P E T A T I S S S V V P E T A T I Y S S V V P E T A T I S P S V V PE T A T I Y P S V V P E T A T I S S R K V P E T A T I Y S R K V P E T AT I S P R K V P E T A T I Y P R K V P E T A T I S S S K V P E T A T I YS S K V P E T A T I S P S K V P E T A T I Y P S K V P E T A T I S S R VA P E T A T I Y S R V A P E T A T I S P R V A P E T A T I Y P R V A P ET A T I S S S V A P E T A T I Y S S V A P E T A T I S P S V A P E T A TI Y P S V A P E T A T I S S R K A P E T A T I Y S R K A P E T A T I S PR K A P E T A T I Y P R K A P E T A T I S S S K A P E T A T I Y S S K AP E T A T I S P S K A P E T A T I Y P S K A P E T A T I S S R V V T Q TA T I Y S R V V T Q T A T I S P R V V T Q T A T I Y P R V V T Q T A T IS S S V V T Q T A T I Y S S V V T Q T A T I S P S V V T Q T A T I Y P SV V T Q T A T I S S R K V T Q T A T I Y S R K V T Q T A T I S P R K V TQ T A T I Y P R K V T Q T A T I S S S K V T Q T A T I Y S S K V T Q T AT I S P S K V T Q T A T I Y P S K V T Q T A T I S S R V A T Q T A T I YS R V A T Q T A T I S P R V A T Q T A T I Y P R V A T Q T A T I S S S VA T Q T A T I Y S S V A T Q T A T I S P S V A T Q T A T I Y P S V A T QT A T I S S R K A T Q T A T I Y S R K A T Q T A T I S P R K A T Q T A TI Y P R K A T Q T A T I S S S K A T Q T A T I Y S S K A T Q T A T I S PS K A T Q T A T I Y P S K A T Q T A T I S S R V V P Q T A T I Y S R V VP Q T A T I S P R V V P Q T A T I Y P R V V P Q T A T I S S S V V P Q TA T I Y S S V V P Q T A T I S P S V V P Q T A T I Y P S V V P Q T A T IS S R K V P Q T A T I Y S R K V P Q T A T I S P R K V P Q T A T I Y P RK V P Q T A T I S S S K V P Q T A T I Y S S K V P Q T A T I S P S K V PQ T A T I Y P S K V P Q T A T I S S R V A P Q T A T I Y S R V A P Q T AT I S P R V A P Q T A T I Y P R V A P Q T A T I S S S V A P Q T A T I YS S V A P Q T A T I S P S V A P Q T A T I Y P S V A P Q T A T I S S R KA P Q T A T I Y S R K A P Q T A T I S P R K A P Q T A T I Y P R K A P QT A T I S S S K A P Q T A T I Y S S K A P Q T A T I S P S K A P Q T A TI Y P S K A P Q T A T I S S R V V T E S A T I Y S R V V T E S A T I S PR V V T E S A T I Y P R V V T E S A T I S S S V V T E S A T I Y S S V VT E S A T I S P S V V T E S A T I Y P S V V T E S A T I S S R K V T E SA T I Y S R K V T E S A T I S P R K V T E S A T I Y P R K V T E S A T IS S S K V T E S A T I Y S S K V T E S A T I S P S K V T E S A T I Y P SK V T E S A T I S S R V A T E S A T I Y S R V A T E S A T I S P R V A TE S A T I Y P R V A T E S A T I S S S V A T E S A T I Y S S V A T E S AT I S P S V A T E S A T I Y P S V A T E S A T I S S R K A T E S A T I YS R K A T E S A T I S P R K A T E S A T I Y P R K A T E S A T I S S S KA T E S A T I Y S S K A T E S A T I S P S K A T E S A T I Y P S K A T ES A T I S S R V V P E S A T I Y S R V V P E S A T I S P R V V P E S A TI Y P R V V P E S A T I S S S V V P E S A T I Y S S V V P E S A T I S PS V V P E S A T I Y P S V V P E S A T I S S R K V P E S A T I Y S R K VP E S A T I S P R K V P E S A T I Y P R K V P E S A T I S S S K V P E SA T I Y S S K V P E S A T I S P S K V P E S A T I Y P S K V P E S A T IS S R V A P E S A T I Y S R V A P E S A T I S P R V A P E S A T I Y P RV A P E S A T I S S S V A P E S A T I Y S S V A P E S A T I S P S V A PE S A T I Y P S V A P E S A T I S S R K A P E S A T I Y S R K A P E S AT I S P R K A P E S A T I Y P R K A P E S A T I S S S K A P E S A T I YS S K A P E S A T I S P S K A P E S A T I Y P S K A P E S A T I S S R VV T Q S A T I Y S R V V T Q S A T I S P R V V T Q S A T I Y P R V V T QS A T I S S S V V T Q S A T I Y S S V V T Q S A T I S P S V V T Q S A TI Y P S V V T Q S A T I S S R K V T Q S A T I Y S R K V T Q S A T I S PR K V T Q S A T I Y P R K V T Q S A T I S S S K V T Q S A T I Y S S K VT Q S A T I S P S K V T Q S A T I Y P S K V T Q S A T I S S R V A T Q SA T I Y S R V A T Q S A T I S P R V A T Q S A T I Y P R V A T Q S A T IS S S V A T Q S A T I Y S S V A T Q S A T I S P S V A T Q S A T I Y P SV A T Q S A T I S S R K A T Q S A T I Y S R K A T Q S A T I S P R K A TQ S A T I Y P R K A T Q S A T I S S S K A T Q S A T I Y S S K A T Q S AT I S P S K A T Q S A T I Y P S K A T Q S A T I S S R V V P Q S A T I YS R V V P Q S A T I S P R V V P Q S A T I Y P R V V P Q S A T I S S S VV P Q S A T I Y S S V V P Q S A T I S P S V V P Q S A T I Y P S V V P QS A T I S S R K V P Q S A T I Y S R K V P Q S A T I S P R K V P Q S A TI Y P R K V P Q S A T I S S S K V P Q S A T I Y S S K V P Q S A T I S PS K V P Q S A T I Y P S K V P Q S A T I S S R V A P Q S A T I Y S R V AP Q S A T I S P R V A P Q S A T I Y P R V A P Q S A T I S S S V A P Q SA T I Y S S V A P Q S A T I S P S V A P Q S A T I Y P S V A P Q S A T IS S R K A P Q S A T I Y S R K A P Q S A T I S P R K A P Q S A T I Y P RK A P Q S A T I S S S K A P Q S A T I Y S S K A P Q S A T I S P S K A PQ S A T I Y P S K A P Q S A T I S S R V V T E T V S I Y S R V V T E T VS I S P R V V T E T V S I Y P R V V T E T V S I S S S V V T E T V S I YS S V V T E T V S I S P S V V T E T V S I Y P S V V T E T V S I S S R KV T E T V S I Y S R K V T E T V S I S P R K V T E T V S I Y P R K V T ET V S I S S S K V T E T V S I Y S S K V T E T V S I S P S K V T E T V SI Y P S K V T E T V S I S S R V A T E T V S I Y S R V A T E T V S I S PR V A T E T V S I Y P R V A T E T V S I S S S V A T E T V S I Y S S V AT E T V S I S P S V A T E T V S I Y P S V A T E T V S I S S R K A T E TV S I Y S R K A T E T V S I S P R K A T E T V S I Y P R K A T E T V S IS S S K A T E T V S I Y S S K A T E T V S I S P S K A T E T V S I Y P SK A T E T V S I S S R V V P E T V S I Y S R V V P E T V S I S P R V V PE T V S I Y P R V V P E T V S I S S S V V P E T V S I Y S S V V P E T VS I S P S V V P E T V S I Y P S V V P E T V S I S S R K V P E T V S I YS R K V P E T V S I S P R K V P E T V S I Y P R K V P E T V S I S S S KV P E T V S I Y S S K V P E T V S I S P S K V P E T V S I Y P S K V P ET V S I S S R V A P E T V S I Y S R V A P E T V S I S P R V A P E T V SI Y P R V A P E T V S I S S S V A P E T V S I Y S S V A P E T V S I S PS V A P E T V S I Y P S V A P E T V S I S S R K A P E T V S I Y S R K AP E T V S I S P R K A P E T V S I Y P R K A P E T V S I S S S K A P E TV S I Y S S K A P E T V S I S P S K A P E T V S I Y P S K A P E T V S IS S R V V T Q T V S I Y S R V V T Q T V S I S P R V V T Q T V S I Y P RV V T Q T V S I S S S V V T Q T V S I Y S S V V T Q T V S I S P S V V TQ T V S I Y P S V V T Q T V S I S S R K V T Q T V S I Y S R K V T Q T VS I S P R K V T Q T V S I Y P R K V T Q T V S I S S S K V T Q T V S I YS S K V T Q T V S I S P S K V T Q T V S I Y P S K V T Q T V S I S S R VA T Q T V S I Y S R V A T Q T V S I S P R V A T Q T V S I Y P R V A T QT V S I S S S V A T Q T V S I Y S S V A T Q T V S I S P S V A T Q T V SI Y P S V A T Q T V S I S S R K A T Q T V S I Y S R K A T Q T V S I S PR K A T Q T V S I Y P R K A T Q T V S I S S S K A T Q T V S I Y S S K AT Q T V S I S P S K A T Q T V S I Y P S K A T Q T V S I S S R V V P Q TV S I Y S R V V P Q T V S I S P R V V P Q T V S I Y P R V V P Q T V S IS S S V V P Q T V S I Y S S V V P Q T V S I S P S V V P Q T V S I Y P SV V P Q T V S I S S R K V P Q T V S I Y S R K V P Q T V S I S P R K V PQ T V S I Y P R K V P Q T V S I S S S K V P Q T V S I Y S S K V P Q T VS I S P S K V P Q T V S I Y P S K V P Q T V S I S S R V A P Q T V S I YS R V A P Q T V S I S P R V A P Q T V S I Y P R V A P Q T V S I S S S VA P Q T V S I Y S S V A P Q T V S I S P S V A P Q T V S I Y P S V A P QT V S I S S R K A P Q T V S I Y S R K A P Q T V S I S P R K A P Q T V SI Y P R K A P Q T V S I S S S K A P Q T V S I Y S S K A P Q T V S I S PS K A P Q T V S I Y P S K A P Q T V S I S S R V V T E S V S I Y S R V VT E S V S I S P R V V T E S V S I Y P R V V T E S V S I S S S V V T E SV S I Y S S V V T E S V S I S P S V V T E S V S I Y P S V V T E S V S IS S R K V T E S V S I Y S R K V T E S V S I S P R K V T E S V S I Y P RK V T E S V S I S S S K V T E S V S I Y S S K V T E S V S I S P S K V TE S V S I Y P S K V T E S V S I S S R V A T E S V S I Y S R V A T E S VS I S P R V A T E S V S I Y P R V A T E S V S I S S S V A T E S V S I YS S V A T E S V S I S P S V A T E S V S I Y P S V A T E S V S I S S R KA T E S V S I Y S R K A T E S V S I S P R K A T E S V S I Y P R K A T ES V S I S S S K A T E S V S I Y S S K A T E S V S I S P S K A T E S V SI Y P S K A T E S V S I S S R V V P E S V S I Y S R V V P E S V S I S PR V V P E S V S I Y P R V V P E S V S I S S S V V P E S V S I Y S S V VP E S V S I S P S V V P E S V S I Y P S V V P E S V S I S S R K V P E SV S I Y S R K V P E S V S I S P R K V P E S V S I Y P R K V P E S V S IS S S K V P E S V S I Y S S K V P E S V S I S P S K V P E S V S I Y P SK V P E S V S I S S R V A P E S V S I Y S R V A P E S V S I S P R V A PE S V S I Y P R V A P E S V S I S S S V A P E S V S I Y S S V A P E S VS I S P S V A P E S V S I Y P S V A P E S V S I S S R K A P E S V S I YS R K A P E S V S I S P R K A P E S V S I Y P R K A P E S V S I S S S KA P E S V S I Y S S K A P E S V S I S P S K A P E S V S I Y P S K A P ES V S I S S R V V T Q S V S I Y S R V V T Q S V S I S P R V V T Q S V SI Y P R V V T Q S V S I S S S V V T Q S V S I Y S S V V T Q S V S I S PS V V T Q S V S I Y P S V V T Q S V S I S S R K V T Q S V S I Y S R K VT Q S V S I S P R K V T Q S V S I Y P R K V T Q S V S I S S S K V T Q SV S I Y S S K V T Q S V S I S P S K V T Q S V S I Y P S K V T Q S V S IS S R V A T Q S V S I Y S R V A T Q S V S I S P R V A T Q S V S I Y P RV A T Q S V S I S S S V A T Q S V S I Y S S V A T Q S V S I S P S V A TQ S V S I Y P S V A T Q S V S I S S R K A T Q S V S I Y S R K A T Q S VS I S P R K A T Q S V S I Y P R K A T Q S V S I S S S K A T Q S V S I YS S K A T Q S V S I S P S K A T Q S V S I Y P S K A T Q S V S I S S R VV P Q S V S I Y S R V V P Q S V S I S P R V V P Q S V S I Y P R V V P QS V S I S S S V V P Q S V S I Y S S V V P Q S V S I S P S V V P Q S V SI Y P S V V P Q S V S I S S R K V P Q S V S I Y S R K V P Q S V S I S PR K V P Q S V S I Y P R K V P Q S V S I S S S K V P Q S V S I Y S S K VP Q S V S I S P S K V P Q S V S I Y P S K V P Q S V S I S S R V A P Q SV S I Y S R V A P Q S V S I S P R V A P Q S V S I Y P R V A P Q S V S IS S S V A P Q S V S I Y S S V A P Q S V S I S P S V A P Q S V S I Y P SV A P Q S V S I S S R K A P Q S V S I Y S R K A P Q S V S I S P R K A PQ S V S I Y P R K A P Q S V S I S S S K A P Q S V S I Y S S K A P Q S VS I S P S K A P Q S V S I Y P S K A P Q S V S I S S R V V T E T A S I YS R V V T E T A S I S P R V V T E T A S I Y P R V V T E T A S I S S S VV T E T A S I Y S S V V T E T A S I S P S V V T E T A S I Y P S V V T ET A S I S S R K V T E T A S I Y S R K V T E T A S I S P R K V T E T A SI Y P R K V T E T A S I S S S K V T E T A S I Y S S K V T E T A S I S PS K V T E T A S I Y P S K V T E T A S I S S R V A T E T A S I Y S R V AT E T A S I S P R V A T E T A S I Y P R V A T E T A S I S S S V A T E TA S I Y S S V A T E T A S I S P S V A T E T A S I Y P S V A T E T A S IS S R K A T E T A S I Y S R K A T E T A S I S P R K A T E T A S I Y P RK A T E T A S I S S S K A T E T A S I Y S S K A T E T A S I S P S K A TE T A S I Y P S K A T E T A S I S S R V V P E T A S I Y S R V V P E T AS I S P R V V P E T A S I Y P R V V P E T A S I S S S V V P E T A S I YS S V V P E T A S I S P S V V P E T A S I Y P S V V P E T A S I S S R KV P E T A S I Y S R K V P E T A S I S P R K V P E T A S I Y P R K V P ET A S I S S S K V P E T A S I Y S S K V P E T A S I S P S K V P E T A SI Y P S K V P E T A S I S S R V A P E T A S I Y S R V A P E T A S I S PR V A P E T A S I Y P R V A P E T A S I S S S V A P E T A S I Y S S V AP E T A S I S P S V A P E T A S I Y P S V A P E T A S I S S R K A P E TA S I Y S R K A P E T A S I S P R K A P E T A S I Y P R K A P E T A S IS S S K A P E T A S I Y S S K A P E T A S I S P S K A P E T A S I Y P SK A P E T A S I S S R V V T Q T A S I Y S R V V T Q T A S I S P R V V TQ T A S I Y P R V V T Q T A S I S S S V V T Q T A S I Y S S V V T Q T AS I S P S V V T Q T A S I Y P S V V T Q T A S I S S R K V T Q T A S I YS R K V T Q T A S I S P R K V T Q T A S I Y P R K V T Q T A S I S S S KV T Q T A S I Y S S K V T Q T A S I S P S K V T Q T A S I Y P S K V T QT A S I S S R V A T Q T A S I Y S R V A T Q T A S I S P R V A T Q T A SI Y P R V A T Q T A S I S S S V A T Q T A S I Y S S V A T Q T A S I S PS V A T Q T A S I Y P S V A T Q T A S I S S R K A T Q T A S I Y S R K AT Q T A S I S P R K A T Q T A S I Y P R K A T Q T A S I S S S K A T Q TA S I Y S S K A T Q T A S I S P S K A T Q T A S I Y P S K A T Q T A S IS S R V V P Q T A S I Y S R V V P Q T A S I S P R V V P Q T A S I Y P RV V P Q T A S I S S S V V P Q T A S I Y S S V V P Q T A S I S P S V V PQ T A S I Y P S V V P Q T A S I S S R K V P Q T A S I Y S R K V P Q T AS I S P R K V P Q T A S I Y P R K V P Q T A S I S S S K V P Q T A S I YS S K V P Q T A S I S P S K V P Q T A S I Y P S K V P Q T A S I S S R VA P Q T A S I Y S R V A P Q T A S I S P R V A P Q T A S I Y P R V A P QT A S I S S S V A P Q T A S I Y S S V A P Q T A S I S P S V A P Q T A SI Y P S V A P Q T A S I S S R K A P Q T A S I Y S R K A P Q T A S I S PR K A P Q T A S I Y P R K A P Q T A S I S S S K A P Q T A S I Y S S K AP Q T A S I S P S K A P Q T A S I Y P S K A P Q T A S I S S R V V T E SA S I Y S R V V T E S A S I S P R V V T E S A S I Y P R V V T E S A S IS S S V V T E S A S I Y S S V V T E S A S I S P S V V T E S A S I Y P SV V T E S A S I S S R K V T E S A S I Y S R K V T E S A S I S P R K V TE S A S I Y P R K V T E S A S I S S S K V T E S A S I Y S S K V T E S AS I S P S K V T E S A S I Y P S K V T E S A S I S S R V A T E S A S I YS R V A T E S A S I S P R V A T E S A S I Y P R V A T E S A S I S S S VA T E S A S I Y S S V A T E S A S I S P S V A T E S A S I Y P S V A T ES A S I S S R K A T E S A S I Y S R K A T E S A S I S P R K A T E S A SI Y P R K A T E S A S I S S S K A T E S A S I Y S S K A T E S A S I S PS K A T E S A S I Y P S K A T E S A S I S S R V V P E S A S I Y S R V VP E S A S I S P R V V P E S A S I Y P R V V P E S A S I S S S V V P E SA S I Y S S V V P E S A S I S P S V V P E S A S I Y P S V V P E S A S IS S R K V P E S A S I Y S R K V P E S A S I S P R K V P E S A S I Y P RK V P E S A S I S S S K V P E S A S I Y S S K V P E S A S I S P S K V PE S A S I Y P S K V P E S A S I S S R V A P E S A S I Y S R V A P E S AS I S P R V A P E S A S I Y P R V A P E S A S I S S S V A P E S A S I YS S V A P E S A S I S P S V A P E S A S I Y P S V A P E S A S I S S R KA P E S A S I Y S R K A P E S A S I S P R K A P E S A S I Y P R K A P ES A S I S S S K A P E S A S I Y S S K A P E S A S I S P S K A P E S A SI Y P S K A P E S A S I S S R V V T Q S A S I Y S R V V T Q S A S I S PR V V T Q S A S I Y P R V V T Q S A S I S S S V V T Q S A S I Y S S V VT Q S A S I S P S V V T Q S A S I Y P S V V T Q S A S I S S R K V T Q SA S I Y S R K V T Q S A S I S P R K V T Q S A S I Y P R K V T Q S A S IS S S K V T Q S A S I Y S S K V T Q S A S I S P S K V T Q S A S I Y P SK V T Q S A S I S S R V A T Q S A S I Y S R V A T Q S A S I S P R V A TQ S A S I Y P R V A T Q S A S I S S S V A T Q S A S I Y S S V A T Q S AS I S P S V A T Q S A S I Y P S V A T Q S A S I S S R K A T Q S A S I YS R K A T Q S A S I S P R K A T Q S A S I Y P R K A T Q S A S I S S S KA T Q S A S I Y S S K A T Q S A S I S P S K A T Q S A S I Y P S K A T QS A S I S S R V V P Q S A S I Y S R V V P Q S A S I S P R V V P Q S A SI Y P R V V P Q S A S I S S S V V P Q S A S I Y S S V V P Q S A S I S PS V V P Q S A S I Y P S V V P Q S A S I S S R K V P Q S A S I Y S R K VP Q S A S I S P R K V P Q S A S I Y P R K V P Q S A S I S S S K V P Q SA S I Y S S K V P Q S A S I S P S K V P Q S A S I Y P S K V P Q S A S IS S R V A P Q S A S I Y S R V A P Q S A S I S P R V A P Q S A S I Y P RV A P Q S A S I S S S V A P Q S A S I Y S S V A P Q S A S I S P S V A PQ S A S I Y P S V A P Q S A S I S S R K A P Q S A S I Y S R K A P Q S AS I S P R K A P Q S A S I Y P R K A P Q S A S I S S S K A P Q S A S I YS S K A P Q S A S I S P S K A P Q S A S I Y P S K A P Q S A S I S S R VV T E T V T V Y S R V V T E T V T V S P R V V T E T V T V Y P R V V T ET V T V S S S V V T E T V T V Y S S V V T E T V T V S P S V V T E T V TV Y P S V V T E T V T V S S R K V T E T V T V Y S R K V T E T V T V S PR K V T E T V T V Y P R K V T E T V T V S S S K V T E T V T V Y S S K VT E T V T V S P S K V T E T V T V Y P S K V T E T V T V S S R V A T E TV T V Y S R V A T E T V T V S P R V A T E T V T V Y P R V A T E T V T VS S S V A T E T V T V Y S S V A T E T V T V S P S V A T E T V T V Y P SV A T E T V T V S S R K A T E T V T V Y S R K A T E T V T V S P R K A TE T V T V Y P R K A T E T V T V S S S K A T E T V T V Y S S K A T E T VT V S P S K A T E T V T V Y P S K A T E T V T V S S R V V P E T V T V YS R V V P E T V T V S P R V V P E T V T V Y P R V V P E T V T V S S S VV P E T V T V Y S S V V P E T V T V S P S V V P E T V T V Y P S V V P ET V T V S S R K V P E T V T V Y S R K V P E T V T V S P R K V P E T V TV Y P R K V P E T V T V S S S K V P E T V T V Y S S K V P E T V T V S PS K V P E T V T V Y P S K V P E T V T V S S R V A P E T V T V Y S R V AP E T V T V S P R V A P E T V T V Y P R V A P E T V T V S S S V A P E TV T V Y S S V A P E T V T V S P S V A P E T V T V Y P S V A P E T V T VS S R K A P E T V T V Y S R K A P E T V T V S P R K A P E T V T V Y P RK A P E T V T V S S S K A P E T V T V Y S S K A P E T V T V S P S K A PE T V T V Y P S K A P E T V T V S S R V V T Q T V T V Y S R V V T Q T VT V S P R V V T Q T V T V Y P R V V T Q T V T V S S S V V T Q T V T V YS S V V T Q T V T V S P S V V T Q T V T V Y P S V V T Q T V T V S S R KV T Q T V T V Y S R K V T Q T V T V S P R K V T Q T V T V Y P R K V T QT V T V S S S K V T Q T V T V Y S S K V T Q T V T V S P S K V T Q T V TV Y P S K V T Q T V T V S S R V A T Q T V T V Y S R V A T Q T V T V S PR V A T Q T V T V Y P R V A T Q T V T V S S S V A T Q T V T V Y S S V AT Q T V T V S P S V A T Q T V T V Y P S V A T Q T V T V S S R K A T Q TV T V Y S R K A T Q T V T V S P R K A T Q T V T V Y P R K A T Q T V T VS S S K A T Q T V T V Y S S K A T Q T V T V S P S K A T Q T V T V Y P SK A T Q T V T V S S R V V P Q T V T V Y S R V V P Q T V T V S P R V V PQ T V T V Y P R V V P Q T V T V S S S V V P Q T V T V Y S S V V P Q T VT V S P S V V P Q T V T V Y P S V V P Q T V T V S S R K V P Q T V T V YS R K V P Q T V T V S P R K V P Q T V T V Y P R K V P Q T V T V S S S KV P Q T V T V Y S S K V P Q T V T V S P S K V P Q T V T V Y P S K V P QT V T V S S R V A P Q T V T V Y S R V A P Q T V T V S P R V A P Q T V TV Y P R V A P Q T V T V S S S V A P Q T V T V Y S S V A P Q T V T V S PS V A P Q T V T V Y P S V A P Q T V T V S S R K A P Q T V T V Y S R K AP Q T V T V S P R K A P Q T V T V Y P R K A P Q T V T V S S S K A P Q TV T V Y S S K A P Q T V T V S P S K A P Q T V T V Y P S K A P Q T V T VS S R V V T E S V T V Y S R V V T E S V T V S P R V V T E S V T V Y P RV V T E S V T V S S S V V T E S V T V Y S S V V T E S V T V S P S V V TE S V T V Y P S V V T E S V T V S S R K V T E S V T V Y S R K V T E S VT V S P R K V T E S V T V Y P R K V T E S V T V S S S K V T E S V T V YS S K V T E S V T V S P S K V T E S V T V Y P S K V T E S V T V S S R VA T E S V T V Y S R V A T E S V T V S P R V A T E S V T V Y P R V A T ES V T V S S S V A T E S V T V Y S S V A T E S V T V S P S V A T E S V TV Y P S V A T E S V T V S S R K A T E S V T V Y S R K A T E S V T V S PR K A T E S V T V Y P R K A T E S V T V S S S K A T E S V T V Y S S K AT E S V T V S P S K A T E S V T V Y P S K A T E S V T V S S R V V P E SV T V Y S R V V P E S V T V S P R V V P E S V T V Y P R V V P E S V T VS S S V V P E S V T V Y S S V V P E S V T V S P S V V P E S V T V Y P SV V P E S V T V S S R K V P E S V T V Y S R K V P E S V T V S P R K V PE S V T V Y P R K V P E S V T V S S S K V P E S V T V Y S S K V P E S VT V S P S K V P E S V T V Y P S K V P E S V T V S S R V A P E S V T V YS R V A P E S V T V S P R V A P E S V T V Y P R V A P E S V T V S S S VA P E S V T V Y S S V A P E S V T V S P S V A P E S V T V Y P S V A P ES V T V S S R K A P E S V T V Y S R K A P E S V T V S P R K A P E S V TV Y P R K A P E S V T V S S S K A P E S V T V Y S S K A P E S V T V S PS K A P E S V T V Y P S K A P E S V T V S S R V V T Q S V T V Y S R V VT Q S V T V S P R V V T Q S V T V Y P R V V T Q S V T V S S S V V T Q SV T V Y S S V V T Q S V T V S P S V V T Q S V T V Y P S V V T Q S V T VS S R K V T Q S V T V Y S R K V T Q S V T V S P R K V T Q S V T V Y P RK V T Q S V T V S S S K V T Q S V T V Y S S K V T Q S V T V S P S K V TQ S V T V Y P S K V T Q S V T V S S R V A T Q S V T V Y S R V A T Q S VT V S P R V A T Q S V T V Y P R V A T Q S V T V S S S V A T Q S V T V YS S V A T Q S V T V S P S V A T Q S V T V Y P S V A T Q S V T V S S R KA T Q S V T V Y S R K A T Q S V T V S P R K A T Q S V T V Y P R K A T QS V T V S S S K A T Q S V T V Y S S K A T Q S V T V S P S K A T Q S V TV Y P S K A T Q S V T V S S R V V P Q S V T V Y S R V V P Q S V T V S PR V V P Q S V T V Y P R V V P Q S V T V S S S V V P Q S V T V Y S S V VP Q S V T V S P S V V P Q S V T V Y P S V V P Q S V T V S S R K V P Q SV T V Y S R K V P Q S V T V S P R K V P Q S V T V Y P R K V P Q S V T VS S S K V P Q S V T V Y S S K V P Q S V T V S P S K V P Q S V T V Y P SK V P Q S V T V S S R V A P Q S V T V Y S R V A P Q S V T V S P R V A PQ S V T V Y P R V A P Q S V T V S S S V A P Q S V T V Y S S V A P Q S VT V S P S V A P Q S V T V Y P S V A P Q S V T V S S R K A P Q S V T V YS R K A P Q S V T V S P R K A P Q S V T V Y P R K A P Q S V T V S S S KA P Q S V T V Y S S K A P Q S V T V S P S K A P Q S V T V Y P S K A P QS V T V S S R V V T E T A T V Y S R V V T E T A T V S P R V V T E T A TV Y P R V V T E T A T V S S S V V T E T A T V Y S S V V T E T A T V S PS V V T E T A T V Y P S V V T E T A T V S S R K V T E T A T V Y S R K VT E T A T V S P R K V T E T A T V Y P R K V T E T A T V S S S K V T E TA T V Y S S K V T E T A T V S P S K V T E T A T V Y P S K V T E T A T VS S R V A T E T A T V Y S R V A T E T A T V S P R V A T E T A T V Y P RV A T E T A T V S S S V A T E T A T V Y S S V A T E T A T V S P S V A TE T A T V Y P S V A T E T A T V S S R K A T E T A T V Y S R K A T E T AT V S P R K A T E T A T V Y P R K A T E T A T V S S S K A T E T A T V YS S K A T E T A T V S P S K A T E T A T V Y P S K A T E T A T V S S R VV P E T A T V Y S R V V P E T A T V S P R V V P E T A T V Y P R V V P ET A T V S S S V V P E T A T V Y S S V V P E T A T V S P S V V P E T A TV Y P S V V P E T A T V S S R K V P E T A T V Y S R K V P E T A T V S PR K V P E T A T V Y P R K V P E T A T V S S S K V P E T A T V Y S S K VP E T A T V S P S K V P E T A T V Y P S K V P E T A T V S S R V A P E TA T V Y S R V A P E T A T V S P R V A P E T A T V Y P R V A P E T A T VS S S V A P E T A T V Y S S V A P E T A T V S P S V A P E T A T V Y P SV A P E T A T V S S R K A P E T A T V Y S R K A P E T A T V S P R K A PE T A T V Y P R K A P E T A T V S S S K A P E T A T V Y S S K A P E T AT V S P S K A P E T A T V Y P S K A P E T A T V S S R V V T Q T A T V YS R V V T Q T A T V S P R V V T Q T A T V Y P R V V T Q T A T V S S S VV T Q T A T V Y S S V V T Q T A T V S P S V V T Q T A T V Y P S V V T QT A T V S S R K V T Q T A T V Y S R K V T Q T A T V S P R K V T Q T A TV Y P R K V T Q T A T V S S S K V T Q T A T V Y S S K V T Q T A T V S PS K V T Q T A T V Y P S K V T Q T A T V S S R V A T Q T A T V Y S R V AT Q T A T V S P R V A T Q T A T V Y P R V A T Q T A T V S S S V A T Q TA T V Y S S V A T Q T A T V S P S V A T Q T A T V Y P S V A T Q T A T VS S R K A T Q T A T V Y S R K A T Q T A T V S P R K A T Q T A T V Y P RK A T Q T A T V S S S K A T Q T A T V Y S S K A T Q T A T V S P S K A TQ T A T V Y P S K A T Q T A T V S S R V V P Q T A T V Y S R V V P Q T AT V S P R V V P Q T A T V Y P R V V P Q T A T V S S S V V P Q T A T V YS S V V P Q T A T V S P S V V P Q T A T V Y P S V V P Q T A T V S S R KV P Q T A T V Y S R K V P Q T A T V S P R K V P Q T A T V Y P R K V P QT A T V S S S K V P Q T A T V Y S S K V P Q T A T V S P S K V P Q T A TV Y P S K V P Q T A T V S S R V A P Q T A T V Y S R V A P Q T A T V S PR V A P Q T A T V Y P R V A P Q T A T V S S S V A P Q T A T V Y S S V AP Q T A T V S P S V A P Q T A T V Y P S V A P Q T A T V S S R K A P Q TA T V Y S R K A P Q T A T V S P R K A P Q T A T V Y P R K A P Q T A T VS S S K A P Q T A T V Y S S K A P Q T A T V S P S K A P Q T A T V Y P SK A P Q T A T V S S R V V T E S A T V Y S R V V T E S A T V S P R V V TE S A T V Y P R V V T E S A T V S S S V V T E S A T V Y S S V V T E S AT V S P S V V T E S A T V Y P S V V T E S A T V S S R K V T E S A T V YS R K V T E S A T V S P R K V T E S A T V Y P R K V T E S A T V S S S KV T E S A T V Y S S K V T E S A T V S P S K V T E S A T V Y P S K V T ES A T V S S R V A T E S A T V Y S R V A T E S A T V S P R V A T E S A TV Y P R V A T E S A T V S S S V A T E S A T V Y S S V A T E S A T V S PS V A T E S A T V Y P S V A T E S A T V S S R K A T E S A T V Y S R K AT E S A T V S P R K A T E S A T V Y P R K A T E S A T V S S S K A T E SA T V Y S S K A T E S A T V S P S K A T E S A T V Y P S K A T E S A T VS S R V V P E S A T V Y S R V V P E S A T V S P R V V P E S A T V Y P RV V P E S A T V S S S V V P E S A T V Y S S V V P E S A T V S P S V V PE S A T V Y P S V V P E S A T V S S R K V P E S A T V Y S R K V P E S AT V S P R K V P E S A T V Y P R K V P E S A T V S S S K V P E S A T V YS S K V P E S A T V S P S K V P E S A T V Y P S K V P E S A T V S S R VA P E S A T V Y S R V A P E S A T V S P R V A P E S A T V Y P R V A P ES A T V S S S V A P E S A T V Y S S V A P E S A T V S P S V A P E S A TV Y P S V A P E S A T V S S R K A P E S A T V Y S R K A P E S A T V S PR K A P E S A T V Y P R K A P E S A T V S S S K A P E S A T V Y S S K AP E S A T V S P S K A P E S A T V Y P S K A P E S A T V S S R V V T Q SA T V Y S R V V T Q S A T V S P R V V T Q S A T V Y P R V V T Q S A T VS S S V V T Q S A T V Y S S V V T Q S A T V S P S V V T Q S A T V Y P SV V T Q S A T V S S R K V T Q S A T V Y S R K V T Q S A T V S P R K V TQ S A T V Y P R K V T Q S A T V S S S K V T Q S A T V Y S S K V T Q S AT V S P S K V T Q S A T V Y P S K V T Q S A T V S S R V A T Q S A T V YS R V A T Q S A T V S P R V A T Q S A T V Y P R V A T Q S A T V S S S VA T Q S A T V Y S S V A T Q S A T V S P S V A T Q S A T V Y P S V A T QS A T V S S R K A T Q S A T V Y S R K A T Q S A T V S P R K A T Q S A TV Y P R K A T Q S A T V S S S K A T Q S A T V Y S S K A T Q S A T V S PS K A T Q S A T V Y P S K A T Q S A T V S S R V V P Q S A T V Y S R V VP Q S A T V S P R V V P Q S A T V Y P R V V P Q S A T V S S S V V P Q SA T V Y S S V V P Q S A T V S P S V V P Q S A T V Y P S V V P Q S A T VS S R K V P Q S A T V Y S R K V P Q S A T V S P R K V P Q S A T V Y P RK V P Q S A T V S S S K V P Q S A T V Y S S K V P Q S A T V S P S K V PQ S A T V Y P S K V P Q S A T V S S R V A P Q S A T V Y S R V A P Q S AT V S P R V A P Q S A T V Y P R V A P Q S A T V S S S V A P Q S A T V YS S V A P Q S A T V S P S V A P Q S A T V Y P S V A P Q S A T V S S R KA P Q S A T V Y S R K A P Q S A T V S P R K A P Q S A T V Y P R K A P QS A T V S S S K A P Q S A T V Y S S K A P Q S A T V S P S K A P Q S A TV Y P S K A P Q S A T V S S R V V T E T V S V Y S R V V T E T V S V S PR V V T E T V S V Y P R V V T E T V S V S S S V V T E T V S V Y S S V VT E T V S V S P S V V T E T V S V Y P S V V T E T V S V S S R K V T E TV S V Y S R K V T E T V S V S P R K V T E T V S V Y P R K V T E T V S VS S S K V T E T V S V Y S S K V T E T V S V S P S K V T E T V S V Y P SK V T E T V S V S S R V A T E T V S V Y S R V A T E T V S V S P R V A TE T V S V Y P R V A T E T V S V S S S V A T E T V S V Y S S V A T E T VS V S P S V A T E T V S V Y P S V A T E T V S V S S R K A T E T V S V YS R K A T E T V S V S P R K A T E T V S V Y P R K A T E T V S V S S S KA T E T V S V Y S S K A T E T V S V S P S K A T E T V S V Y P S K A T ET V S V S S R V V P E T V S V Y S R V V P E T V S V S P R V V P E T V SV Y P R V V P E T V S V S S S V V P E T V S V Y S S V V P E T V S V S PS V V P E T V S V Y P S V V P E T V S V S S R K V P E T V S V Y S R K VP E T V S V S P R K V P E T V S V Y P R K V P E T V S V S S S K V P E TV S V Y S S K V P E T V S V S P S K V P E T V S V Y P S K V P E T V S VS S R V A P E T V S V Y S R V A P E T V S V S P R V A P E T V S V Y P RV A P E T V S V S S S V A P E T V S V Y S S V A P E T V S V S P S V A PE T V S V Y P S V A P E T V S V S S R K A P E T V S V Y S R K A P E T VS V S P R K A P E T V S V Y P R K A P E T V S V S S S K A P E T V S V YS S K A P E T V S V S P S K A P E T V S V Y P S K A P E T V S V S S R VV T Q T V S V Y S R V V T Q T V S V S P R V V T Q T V S V Y P R V V T QT V S V S S S V V T Q T V S V Y S S V V T Q T V S V S P S V V T Q T V SV Y P S V V T Q T V S V S S R K V T Q T V S V Y S R K V T Q T V S V S PR K V T Q T V S V Y P R K V T Q T V S V S S S K V T Q T V S V Y S S K VT Q T V S V S P S K V T Q T V S V Y P S K V T Q T V S V S S R V A T Q TV S V Y S R V A T Q T V S V S P R V A T Q T V S V Y P R V A T Q T V S VS S S V A T Q T V S V Y S S V A T Q T V S V S P S V A T Q T V S V Y P SV A T Q T V S V S S R K A T Q T V S V Y S R K A T Q T V S V S P R K A TQ T V S V Y P R K A T Q T V S V S S S K A T Q T V S V Y S S K A T Q T VS V S P S K A T Q T V S V Y P S K A T Q T V S V S S R V V P Q T V S V YS R V V P Q T V S V S P R V V P Q T V S V Y P R V V P Q T V S V S S S VV P Q T V S V Y S S V V P Q T V S V S P S V V P Q T V S V Y P S V V P QT V S V S S R K V P Q T V S V Y S R K V P Q T V S V S P R K V P Q T V SV Y P R K V P Q T V S V S S S K V P Q T V S V Y S S K V P Q T V S V S PS K V P Q T V S V Y P S K V P Q T V S V S S R V A P Q T V S V Y S R V AP Q T V S V S P R V A P Q T V S V Y P R V A P Q T V S V S S S V A P Q TV S V Y S S V A P Q T V S V S P S V A P Q T V S V Y P S V A P Q T V S VS S R K A P Q T V S V Y S R K A P Q T V S V S P R K A P Q T V S V Y P RK A P Q T V S V S S S K A P Q T V S V Y S S K A P Q T V S V S P S K A PQ T V S V Y P S K A P Q T V S V S S R V V T E S V S V Y S R V V T E S VS V S P R V V T E S V S V Y P R V V T E S V S V S S S V V T E S V S V YS S V V T E S V S V S P S V V T E S V S V Y P S V V T E S V S V S S R KV T E S V S V Y S R K V T E S V S V S P R K V T E S V S V Y P R K V T ES V S V S S S K V T E S V S V Y S S K V T E S V S V S P S K V T E S V SV Y P S K V T E S V S V S S R V A T E S V S V Y S R V A T E S V S V S PR V A T E S V S V Y P R V A T E S V S V S S S V A T E S V S V Y S S V AT E S V S V S P S V A T E S V S V Y P S V A T E S V S V S S R K A T E SV S V Y S R K A T E S V S V S P R K A T E S V S V Y P R K A T E S V S VS S S K A T E S V S V Y S S K A T E S V S V S P S K A T E S V S V Y P SK A T E S V S V S S R V V P E S V S V Y S R V V P E S V S V S P R V V PE S V S V Y P R V V P E S V S V S S S V V P E S V S V Y S S V V P E S VS V S P S V V P E S V S V Y P S V V P E S V S V S S R K V P E S V S V YS R K V P E S V S V S P R K V P E S V S V Y P R K V P E S V S V S S S KV P E S V S V Y S S K V P E S V S V S P S K V P E S V S V Y P S K V P ES V S V S S R V A P E S V S V Y S R V A P E S V S V S P R V A P E S V SV Y P R V A P E S V S V S S S V A P E S V S V Y S S V A P E S V S V S PS V A P E S V S V Y P S V A P E S V S V S S R K A P E S V S V Y S R K AP E S V S V S P R K A P E S V S V Y P R K A P E S V S V S S S K A P E SV S V Y S S K A P E S V S V S P S K A P E S V S V Y P S K A P E S V S VS S R V V T Q S V S V Y S R V V T Q S V S V S P R V V T Q S V S V Y P RV V T Q S V S V S S S V V T Q S V S V Y S S V V T Q S V S V S P S V V TQ S V S V Y P S V V T Q S V S V S S R K V T Q S V S V Y S R K V T Q S VS V S P R K V T Q S V S V Y P R K V T Q S V S V S S S K V T Q S V S V YS S K V T Q S V S V S P S K V T Q S V S V Y P S K V T Q S V S V S S R VA T Q S V S V Y S R V A T Q S V S V S P R V A T Q S V S V Y P R V A T QS V S V S S S V A T Q S V S V Y S S V A T Q S V S V S P S V A T Q S V SV Y P S V A T Q S V S V S S R K A T Q S V S V Y S R K A T Q S V S V S PR K A T Q S V S V Y P R K A T Q S V S V S S S K A T Q S V S V Y S S K AT Q S V S V S P S K A T Q S V S V Y P S K A T Q S V S V S S R V V P Q SV S V Y S R V V P Q S V S V S P R V V P Q S V S V Y P R V V P Q S V S VS S S V V P Q S V S V Y S S V V P Q S V S V S P S V V P Q S V S V Y P SV V P Q S V S V S S R K V P Q S V S V Y S R K V P Q S V S V S P R K V PQ S V S V Y P R K V P Q S V S V S S S K V P Q S V S V Y S S K V P Q S VS V S P S K V P Q S V S V Y P S K V P Q S V S V S S R V A P Q S V S V YS R V A P Q S V S V S P R V A P Q S V S V Y P R V A P Q S V S V S S S VA P Q S V S V Y S S V A P Q S V S V S P S V A P Q S V S V Y P S V A P QS V S V S S R K A P Q S V S V Y S R K A P Q S V S V S P R K A P Q S V SV Y P R K A P Q S V S V S S S K A P Q S V S V Y S S K A P Q S V S V S PS K A P Q S V S V Y P S K A P Q S V S V S S R V V T E T A S V Y S R V VT E T A S V S P R V V T E T A S V Y P R V V T E T A S V S S S V V T E TA S V Y S S V V T E T A S V S P S V V T E T A S V Y P S V V T E T A S VS S R K V T E T A S V Y S R K V T E T A S V S P R K V T E T A S V Y P RK V T E T A S V S S S K V T E T A S V Y S S K V T E T A S V S P S K V TE T A S V Y P S K V T E T A S V S S R V A T E T A S V Y S R V A T E T AS V S P R V A T E T A S V Y P R V A T E T A S V S S S V A T E T A S V YS S V A T E T A S V S P S V A T E T A S V Y P S V A T E T A S V S S R KA T E T A S V Y S R K A T E T A S V S P R K A T E T A S V Y P R K A T ET A S V S S S K A T E T A S V Y S S K A T E T A S V S P S K A T E T A SV Y P S K A T E T A S V S S R V V P E T A S V Y S R V V P E T A S V S PR V V P E T A S V Y P R V V P E T A S V S S S V V P E T A S V Y S S V VP E T A S V S P S V V P E T A S V Y P S V V P E T A S V S S R K V P E TA S V Y S R K V P E T A S V S P R K V P E T A S V Y P R K V P E T A S VS S S K V P E T A S V Y S S K V P E T A S V S P S K V P E T A S V Y P SK V P E T A S V S S R V A P E T A S V Y S R V A P E T A S V S P R V A PE T A S V Y P R V A P E T A S V S S S V A P E T A S V Y S S V A P E T AS V S P S V A P E T A S V Y P S V A P E T A S V S S R K A P E T A S V YS R K A P E T A S V S P R K A P E T A S V Y P R K A P E T A S V S S S KA P E T A S V Y S S K A P E T A S V S P S K A P E T A S V Y P S K A P ET A S V S S R V V T Q T A S V Y S R V V T Q T A S V S P R V V T Q T A SV Y P R V V T Q T A S V S S S V V T Q T A S V Y S S V V T Q T A S V S PS V V T Q T A S V Y P S V V T Q T A S V S S R K V T Q T A S V Y S R K VT Q T A S V S P R K V T Q T A S V Y P R K V T Q T A S V S S S K V T Q TA S V Y S S K V T Q T A S V S P S K V T Q T A S V Y P S K V T Q T A S VS S R V A T Q T A S V Y S R V A T Q T A S V S P R V A T Q T A S V Y P RV A T Q T A S V S S S V A T Q T A S V Y S S V A T Q T A S V S P S V A TQ T A S V Y P S V A T Q T A S V S S R K A T Q T A S V Y S R K A T Q T AS V S P R K A T Q T A S V Y P R K A T Q T A S V S S S K A T Q T A S V YS S K A T Q T A S V S P S K A T Q T A S V Y P S K A T Q T A S V S S R VV P Q T A S V Y S R V V P Q T A S V S P R V V P Q T A S V Y P R V V P QT A S V S S S V V P Q T A S V Y S S V V P Q T A S V S P S V V P Q T A SV Y P S V V P Q T A S V S S R K V P Q T A S V Y S R K V P Q T A S V S PR K V P Q T A S V Y P R K V P Q T A S V S S S K V P Q T A S V Y S S K VP Q T A S V S P S K V P Q T A S V Y P S K V P Q T A S V S S R V A P Q TA S V Y S R V A P Q T A S V S P R V A P Q T A S V Y P R V A P Q T A S VS S S V A P Q T A S V Y S S V A P Q T A S V S P S V A P Q T A S V Y P SV A P Q T A S V S S R K A P Q T A S V Y S R K A P Q T A S V S P R K A PQ T A S V Y P R K A P Q T A S V S S S K A P Q T A S V Y S S K A P Q T AS V S P S K A P Q T A S V Y P S K A P Q T A S V S S R V V T E S A S V YS R V V T E S A S V S P R V V T E S A S V Y P R V V T E S A S V S S S VV T E S A S V Y S S V V T E S A S V S P S V V T E S A S V Y P S V V T ES A S V S S R K V T E S A S V Y S R K V T E S A S V S P R K V T E S A SV Y P R K V T E S A S V S S S K V T E S A S V Y S S K V T E S A S V S PS K V T E S A S V Y P S K V T E S A S V S S R V A T E S A S V Y S R V AT E S A S V S P R V A T E S A S V Y P R V A T E S A S V S S S V A T E SA S V Y S S V A T E S A S V S P S V A T E S A S V Y P S V A T E S A S VS S R K A T E S A S V Y S R K A T E S A S V S P R K A T E S A S V Y P RK A T E S A S V S S S K A T E S A S V Y S S K A T E S A S V S P S K A TE S A S V Y P S K A T E S A S V S S R V V P E S A S V Y S R V V P E S AS V S P R V V P E S A S V Y P R V V P E S A S V S S S V V P E S A S V YS S V V P E S A S V S P S V V P E S A S V Y P S V V P E S A S V S S R KV P E S A S V Y S R K V P E S A S V S P R K V P E S A S V Y P R K V P ES A S V S S S K V P E S A S V Y S S K V P E S A S V S P S K V P E S A SV Y P S K V P E S A S V S S R V A P E S A S V Y S R V A P E S A S V S PR V A P E S A S V Y P R V A P E S A S V S S S V A P E S A S V Y S S V AP E S A S V S P S V A P E S A S V Y P S V A P E S A S V S S R K A P E SA S V Y S R K A P E S A S V S P R K A P E S A S V Y P R K A P E S A S VS S S K A P E S A S V Y S S K A P E S A S V S P S K A P E S A S V Y P SK A P E S A S V S S R V V T Q S A S V Y S R V V T Q S A S V S P R V V TQ S A S V Y P R V V T Q S A S V S S S V V T Q S A S V Y S S V V T Q S AS V S P S V V T Q S A S V Y P S V V T Q S A S V S S R K V T Q S A S V YS R K V T Q S A S V S P R K V T Q S A S V Y P R K V T Q S A S V S S S KV T Q S A S V Y S S K V T Q S A S V S P S K V T Q S A S V Y P S K V T QS A S V S S R V A T Q S A S V Y S R V A T Q S A S V S P R V A T Q S A SV Y P R V A T Q S A S V S S S V A T Q S A S V Y S S V A T Q S A S V S PS V A T Q S A S V Y P S V A T Q S A S V S S R K A T Q S A S V Y S R K AT Q S A S V S P R K A T Q S A S V Y P R K A T Q S A S V S S S K A T Q SA S V Y S S K A T Q S A S V S P S K A T Q S A S V Y P S K A T Q S A S VS S R V V P Q S A S V Y S R V V P Q S A S V S P R V V P Q S A S V Y P RV V P Q S A S V S S S V V P Q S A S V Y S S V V P Q S A S V S P S V V PQ S A S V Y P S V V P Q S A S V S S R K V P Q S A S V Y S R K V P Q S AS V S P R K V P Q S A S V Y P R K V P Q S A S V S S S K V P Q S A S V YS S K V P Q S A S V S P S K V P Q S A S V Y P S K V P Q S A S V S S R VA P Q S A S V Y S R V A P Q S A S V S P R V A P Q S A S V Y P R V A P QS A S V S S S V A P Q S A S V Y S S V A P Q S A S V S P S V A P Q S A SV Y P S V A P Q S A S V S S R K A P Q S A S V Y S R K A P Q S A S V S PR K A P Q S A S V Y P R K A P Q S A S V S S S K A P Q S A S V Y S S K AP Q S A S V S P S K A P Q S A S V Y P S K A P Q S A S V

A further embodiment In some embodiments of the invention, the targetedbinding agent or antibody comprises a sequence comprising SEQ ID NO.: 8.In certain embodiments, SEQ ID NO.: 24 comprises any one of thecombinations of germline and non-germline residues indicated by each rowof Table 12. In some embodiments, SEQ ID NO: 24 comprises any one, anytwo, any three, any four, any five, any six, any seven, any eight, anynine, any ten, any eleven, or all eleven of the germline residues asindicated in Table 12. In certain embodiments, SEQ ID NO.: 24 comprisesany one of the unique combinations of germline and non-germline residuesindicated by each row of Table 12. In other embodiments, the targetedbinding agent or antibody is derived from a germline sequence with VL,3r and JL2 domains, wherein one or more residues has been mutated toyield the corresponding germline residue at that position. In certainembodiments, SEQ ID NO.: 24 can comprise further modifications thatinclude removing structural liabilities. For example, SEQ ID NO.: 24 cancomprise any one of the unique combinations of germline and non-germlineresidues indicated by each row of Table 12 and further include themutation of C33 to a S. An example is 9G8 VLOP1 where the light chainhas been mutated to remove the structural liability at C33 and furthermutated back to the germline sequence at position 7 where the S has beenmutated to a P and V at position 79 has been mutated to an A. Anotherexample is 9G8 VLOP2 where C33 has been mutated to S, S at position 2has been mutated to an Y, S at position 7 has been mutated to a P, R atposition 19 has been mutated to an S, T at position 39 has been mutatedto an P, and V at position 79 has been mutated to an A. See specificallyTable 13.

The skilled person will be aware that there are alternative methods ofdefining CDR boundaries. All CDR boundaries in Table 2 and 13 aredefined according to the Kabat definition.

TABLE 13 SEQ ID NO Chain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 75 21H3 QVQLVQSGAEVKKP NYGIT WVRQAPGQ WISAYNGNTN RVTMTTDTSTSTAYMEL DRVPRIPVTTWGQGTMVTVSS VHOP GASVKVSCKASGYT GLEWMG YAQKLQD RSLRSDDTAVYYCAR EAFDI FT50 21H3 QSVLTQPPSASGTP SGSSSNIGSYF WYQQLPGT RNNQRPS GVPDRFSGSESGTSASLAAWDDSLSGH FGGGTKLTVL VLOP1 GQRVTISC VY APKLLIY AISGLRSEDEADYYC WV(corre- spond-  ing to 21H3RK VL) 76 21H3  QSVLTQPPSASGTP SGSSSNIGSYFWYQQLPGT RNNQRPS GVPDRFSGSKSGTSASL AAWDDSLSGH FGGGTKLTVL VLOP2 GQRVTISCVY APKLLIY AISGLRSEDEADYYC WV 77 2H10  QVQLVESGGGVVQP RHGMH WVRQAPGKVVWFDGSNIY RFTISRDNSKNTLYLQM DSRIAAADY WGQGTLVTVSS VHOP GRSLRLSCAASGFTGLEWVA YADSVKG NSLRAEDTAVYYCAR FS 78 2H10  SYELTQPPSVSVSP SGDKLGDKYVSWYQQKPGQ QESKRPS GIPERFSGSSSGNTATL QTWDSSLVV FGGGTKLTVL VLOP1 GQTVSITCSPVLVIY TISGTQAMDEADYYC 79 2H10  SYELTQPPSVSVSP SGDKLGDKYVS WYQQKPGQQESKRPS GIPERFSGSNSGNTATL QTWDSSLVV FGGGTKLTVL VLOP2 GQTASITC SPVLVIYTISGTQAMDEADYYC 80 9G8  QLQLQESGPGLVKP SSSSY WGWIRQPP SIYYSGSTYYRVSISVDTSKNQFSLKL QGYGGHPDVF WGQGTMVTVSS VHOP1 SETLSLSCTVSGGS GKGLEWIGSPSLKS SSVTAADTAVYFCAR DI IS 81 9G8  QLQLQESGPGLVKP SSSSY WGWIRQPPSIYYSGSTYY RVTISVDTSKNQFSLKL QGYGGHPDVF WGQGTMVTVSS VHOP2 SETLSLTCTVSGGSGKGLEWIG SPSLKS SSVTAADTAVYYCAR DI IS 82 9G8  SSELTQPPSVSVSP SGDKLGDVYVSWYQQKTGQ EDTKRPS GIPERFSGSNSGNTATL QAWDSTTAVI FGGGTKLTVL VLOP1 GQTARITCSPVLVIY TISGTQAMDEADYYC 83 9G8  SYELTQSPSVSVSP SGDKLGDVYVA WYQQKPGQEDTKRPS GIPERFSGSNSGNTATL QAWDSTTAVI FGGGTKLTVL VLOP2 GQTASITC S SPVLVIYTISGTQAMDEADYYC

Example 6 Potency Determination of Dll4 Antibodies to InhibitNotch1-DLL4 Receptor-Ligand Binding

To discriminate between the purified antibodies based upon their abilityto prevent the interaction between full-length recombinant DLL4 andsoluble Notch1/Fc, the following assays were performed. Transfected anduntransfected 239T cells were reconstituted in PBS containing 2% FCS and5000 transfected and 17500 non-transfected cells were plated in 30 μlinto wells of a 384-well tissue culture plate (Corning Costar, catalog#3712). Subsequently, 30 μl of purified antibodies, serial diluted from5 μg/ml hybridoma supernatant was added and plates were incubated at 4°C. for 1 h, at which time 20 μl of 100 ng/ml Alexa-647 labeled humanNotch1/Fc was added. After a further 3 h incubation at 4° C., the amountof bound Notch1/Fc was determined by reading the fluorescence in eachwell using an FMAT 8200 instrument (Applied Biosystems). Data for 12purified antibodies is shown in Table 14, which shows the ability ofpurified antibodies to inhibit is interactions between recombinantfull-length human DLL4 and Notch1/Fc chimera.

In addition similar experiments were performed and quantified using aFACSCalibur (BD Biosciences) instrument. For these experiments, parental293T cells or cells transiently transfected with human DLL4 werereconstituted in PBS containing 2% FCS and added at a concentration of25000 cells/well to wells containing purified antibodies at a finalconcentration of 10 μg/ml, 1 μg/ml or 0.1 μg/ml. After incubation for 1h at 4° C., Alexa-647 labeled human Notch1/Fc was added at a finalconcentration of 227 ng/ml and plates were incubated for 2 h at 4° C.Following washing with PBS containing 2% FCS, the amount of boundNotch1/Fc was determined by reading the fluorescence in each well usinga FACSCalibur instrument. Under these conditions, the ability of the 20purified antibodies to inhibit DLL4-Notch1 interactions was similar tothat observed using the FMAT instrument (data not shown). Similarresults were also obtained when experiments were performed in an ELISAformat using soluble human DLL4 and soluble human Notch1 (data notshown).

Further experiments were performed on selected antibodies using HEK293cells that were stably transfected with either human, mouse orcynomolgus monkey DLL4 using retroviral constructs. In theseexperiments, anti-DLL4 antibodies (final concentration: 10-0.01 μg/ml)diluted in PBS containing 2% FCS were added to DLL4-expressing HEK 293cells (50000 cells/well diluted in PBS containing 2% FCS) and incubatedfor 1 h at 4° C. Subsequently, Alexa-647 labeled human, rat or APClabeled mouse Notch1/Fc (e.g. R&D Systems, catalog #3647-TK-050,1057-TK-050, 5267-TK-050, respectively) was added at a finalconcentration of 0.01-0.5 μg/ml and plates were incubated for a further2 h at 4° C. prior to washing and reading on a FACSCalibur instrument.Table 15 shows the ability of purified antibodies of different isotypesto inhibit interactions between recombinant full length human,cynomolgus monkey and mouse DLL4 and 0.1 or 0.25 μg/ml human, rat or 0.5μg/ml mouse Notch1/Fc chimera as determined by FACS analysis.

TABLE 14 % % % % % % % inhi- inhi- inhi- inhi- inhi- inhi- inhi- bitionbition bition bition bition bition bition Anti- IC₅₀ 16.7 5.6 1.9 0.620.21 0.070 0.020 body (nM) nM nM nM nM nM nM nM 1D4 2.00 123 124 58 13 5−2 7 1E4 0.99 120 129 91 46 21 24 −7 4B4 0.72 136 127 95 65 23 21 142H10 2.05 111 119 45 27 14 −3 6 3A7 2.94 104 82 39 19 −1 7 −1 4B3 3.33117 89 40 20 −3 5 −1 9G8 1.88 121 125 68 22 33 14 10 12A1 1.48 101 11988 15 9 −18 6 17F3 1.81 115 121 69 29 14 25 14 21F7 1.95 104 116 35 −227 17 11 20G8 2.02 115 114 29 −14 −4 3 −5 21H3 1.73 124 115 68 9 25 −2−17

TABLE 15 Species Mouse (rat Notch1 unless otherwise Human Cyno stated)IC₅₀ ± S.D. IC₅₀ ± S.D. IC₅₀ ± S.D. Antibody Isotype (nM) (nM) (nM) 21H3IgG1 0.57 ± 0.24 0.46 N.T. IgG2 0.73 ± 0.21 N.T. 13.53 IgG4  1.59 ±0.62* N.T. N.T. 21H3RK IgG1 0.58 ± 0.26 0.44 ± 0.10 42 (mouse Notch1)IgG2 0.75 ± 0.12 N.T. 15.61 4B4 IgG1 0.85 ± 0.21 0.50 N.T. IgG2 1.19 ±0.23 N.T. >67 IgG4  1.62 ± 0.64* N.T. N.T. *concentration of humanNotch1/Fc 0.25 μg/ml. N.T. = not tested

Example 7 Cross Reactivity of Dll4 Antibodies to Human Jagged1 and DLL1

The ability of the purified antibodies to bind to human Jagged1 and Dll1was determined by FACS analysis. Briefly, 293T cells were eithermock-transfected or transiently transfected with either human Jagged1(accession #: NM_(—)000214) or Dll1 (accession #: NM_(—)005618) usingLipofectamine 2000. Cells were resuspended in PBS containing 2% FCS andseeded at 50000 cells/well into V-bottomed plates. Anti-DLL4 antibodiesdiluted in PBS containing 2% FCS were added at a final concentration of5 or 15 μg/ml and plates were incubated for 1 h at 4° C. After washingwith PBS containing 2% FCS, secondary antibody (Goat anti-human Fc Cy5,Jackson Immunoresearch, catalog #109-175-098, 5 μg/ml) and 7-AAD (5μg/ml) were added and plates were incubated for 15 min at 4° C. beforebeing washed again with PBS containing 2% FCS and being read on aFACSCalibur instrument. Mouse anti-human Jagged 1 antibody (R&D systems,catalog #mAB1277, detected with anti-mouse Fc Cy5 secondary antibody (5μg/ml), Jackson Immunoresearch catalog #115-175-164), human Notch3/Fcchimera (R&D systems, catalog #1559-NT-050, detected with Goatanti-human Fc Cy5 antibody described above)) or Goat anti-human Dll1antibody (R&D systems cat #AF1818, detected with anti-goat Fc Cy5,Jackson Immunoresearch, catalog #305-175-046 (5 μg/ml)) were used ascontrols to confirm transfection. Data was analysed by comparing theshift in geometric mean fluorescence in the mock-transfected cells tothat observed in the Jagged1- or Dll1-transfected cells and is shown inTables 16 and 17. Table 16 shows ability of purified antibodies (5μg/ml) to bind to 293T cells transiently transfected with human Jagged1.Table 17 shows the ability of purified antibodies (15 μg/ml) to bind to293T cells transiently transfected with human Dll1. Additional FACSbinding studies using 21H3RK, 4B4, 9G8 or 2H10 at concentrations up to300 μg/ml demonstrated minimal (<2.5-fold over background) binding toHEK-293 cells that stably overexpressed either human Jagged1 or humanDll1.

TABLE 16 Jagged1/293T Mock/293T Antibody X Geo mean X Geo meanJagged1/mock ratio 1D4 16.8 15.8 1.01 1E4 16.9 14.9 1.13 4B4 16.8 15.41.09 2H10 9.28 7.36 1.26 3A7 16.4 15.0 1.10 4B3 18.7 18.1 1.04 9G8 17.416.2 1.07 12A1 14.4 12.9 1.11 17F3 16.3 15.2 1.08 21F7 13.8 13.7 1.0120G8 16.0 14.3 1.12 21H3 15.5 13.8 1.12 N3 Fc 5 μg/ml 51.0 13.2 3.87 N3Fc 1 μg/ml 17.9 6.23 2.88 N3 Fc 0.2 μg/ml 8.60 4.39 1.96 N3 Fc 0 μg/ml4.13 3.78 1.09 Jagged1 Ab 5 μg/ml 196 45.6 4.29 Jagged1 Ab 1 μg/ml 11930.2 3.94 Jagged1 Ab 0.2 μg/ml 50.7 15.0 3.38 Jagged 1 Ab 0 μg/ml 4.614.13 1.12

TABLE 17 Dl11/293T Mock/293T Antibody X Geo mean X Geo mean Dl11/mockratio 1D4 35.3 32.2 1.09 1E4 31.7 27.7 1.15 4B4 25.9 21.1 1.23 2H10 44.140.1 1.10 3A7 36.8 34.6 1.06 4B3 30.6 26.0 1.17 9G8 32.3 26.3 1.23 12A147.0 42.1 1.12 17F3 47.0 41.3 1.14 21F7 30.2 26.8 1.13 20G8 40.0 30.01.34 21H3 32.4 27.9 1.16 Dl11 Ab 5 μg/ml 1840 239 7.68 Dl11 Ab 1 μg/ml1240 197 6.33 Dl11 Ab 0.2 μg/ml 565 81.4 6.94

Example 8 Determination of the Effects of Dll4 Antibodies onDLL4-Mediated HUVEC Proliferation

The ability of DLL4 antibodies to block DLL4-stimulated inhibition ofHUVEC proliferation was evaluated. DLL4 extracellular domain (R&Dsystems, catalog #1506-D4/CF) was prepared as a 50 μg/ml stock in PBScontaining 0.1% BSA. Following dilution to 1 μg/ml in bicarbonate buffer(Sigma #C3041-50CAP), 100 μl/well was added to black walled 96 wellplates (Perkin Elmer, catalog #6005182) and plates were incubatedovernight at 4° C. Control wells were also mock coated with PBScontaining 0.1% BSA. After washing with PBS, 100 μl HUVEC cells at aconcentration of 4E4 cells/ml in MCDB 113 (Gibco catalog #10372)containing 10% FCS and 2 mM glutamine was added to each well.Immediately afterwards, serially diluted anti-DLL4 antibodies (20-0.027μg/ml) were added to DLL4/mock coated wells in triplicate and cells wereincubated for 96 h at 37° C./5% CO₂. After this incubation, 15 μl ofCell Counting Kit 8 (CCK8, NBS, catalog #CK-04-11) was added to eachwell and plates were incubated for 4 h at 37° C./5% CO₂. To determinerelative cell number in each well, absorbance at 450 nm was measured ona platereader (Tecan Ultra). The effects of the anti-DLL4 antibodies aredetailed in Table 18. In addition, 4B4, 21H3 and 21H3RK were alsoeffective inhibitors of the DLL4 mediated effects when formatted as IgG1antibodies (FIG. 1).

In addition, the abilities of the anti-DLL4 antibodies (10 μg/ml) toinhibit Notch signaling were evaluated via Western blot. Briefly,DLL4-His (R&D systems, catalog #1506-D4-050/CF) was diluted to 50 μg/mlin PBS containing 0.1% BSA. This solution was then further diluted to 1μg/ml in 50 mM bicarbonate buffer, pH 9.6 (Sigma catalog #C-3041) and 1ml per well added to 12 well plates and incubated overnight at 4° C.Additional wells not containing DLL4 were mock-coated using the sameprocedure. After washing with PBS, HUVEC cells prepared in MCDB131medium were seeded at 12000 cells/well. Immediately after seeding, theappropriate treatment (e.g. anti-DLL4 antibodies, 10 μg/ml) was addedand plates were incubated for 24 h at 37° C./5% CO₂. After theincubation was completed, cells were harvested in RIPA buffer. 4× samplebuffer containing B-mercaptoethanol and bromophenol blue was then addedand samples were boiled for 5 min at 70° C. prior to loading onto 4-12%NuPAGE gels in MOPS buffer (Invitrogen catalog #NP0001). Followingelectrophoretic transfer to nitrocellulose, blots were blocked for 1 hin PBST containing 5% milk followed by incubation with either cleavedanti-Notch1 (Cell signaling technology, catalog #2421) or GAPDH(Advanced Immunochemical, clone 6C5, catalog #2-RGM2) antibodies at1:1000 or 1:10,000 dilutions, respectively in PBS containing 5% milk.After incubation on an orbital shaker overnight at 4° C., blots werewashed with PBST and incubated with anti-mouse-HRP secondary antibody(Cell Signaling Technology, catalog #7072) at a concentration of 1:2000in PBST containing 5% milk for 1 h at RT. After washing with PBST, blotswere developed using Pierce Pico (catalog #34080; GAPDH) or Femto(catalog #34075; cleaved Notch1) substrate reagents and results analysedon a ChemiGenius instrument. The results obtained from these studiesdemonstrate that the anti-DLL4 antibodies can block DLL4-stimulatedNotch signaling in HUVEC cells (data not shown).

TABLE 18 N = 1 N = 2 AVERAGE % inhi- % inhi- % inhi- % inhi- % inhi- %inhi- bition bition bition bition bition bition Iso- 1 μg/ 10 μg/ 1 μg/10 μg/ 1 μg/ 10 μg/ Ab type ml ml ml ml ml ml 1D4 IgG4 43 73 46 71 45 721E4 IgG2 73 81 75 82 74 82 4B4 IgG4 101 102 84 92 93 97 2H10 IgG4 98 11087 95 93 103 3A7 IgG4 50 74 80 106 65 90 4B3 IgG4 20 38 41 49 31 44 9G8IgG4 52 72 71 82 62 77 12A1 IgG4 50 62 62 85 56 74 17F3 IgG2 24 48 35 5630 52 21F7 IgG2 30 48 N.T. N.T. N/A N/A 20G8 IgG4 94 103 75 93 85 9821H3 IgG4 86 88 99 89 93 89 Control IgG2 18 11 0 9 9 10 Control IgG4 146 8 −3 11 2 N/A = Not applicable. N.T. = Not tested

Example 9 Effects of DLL4 Antibodies on HUVEC Tube Formation In Vitro

DLL4 inhibitory antibodies were tested for the ability to reduceendothelial cell tube formation in an in vitro co-culture assay (e.g.,TCS Cell Works Cat no. ZHA-1000). Human Umbilical Vein Endothelial Cells(HUVECs) and human diploid fibroblasts were either obtained asco-cultures in 24 well plates (TCS Cell works Cat no. ZHA-1000) orplates were prepared as follows: 24 well tissue culture plates werecoated with collagen (1:10 dilution in distilled water; Sigma, catalog#C8919) at 37° C./5% CO₂ for 4 h. After washing with PBS, fibroblasts(e.g. Promocell #C-12300) were added at 15000 cells/well in FGM(Promocell #C-23010). After incubating for 3 days at 37° C./5% CO₂, themedia was removed from the plate and HUVEC cells at 30,000 cells/well inEGM2 (Promocell #C-22111) were added. After incubating for a further 4days at 37° C./5% CO₂, plates were considered ready for use and this wasconsidered day 1 for the assay. DLL4 blocking antibodies were introducedto the cultures on day 1 and at regular intervals over an 11-day periodat concentrations ranging from 20 to 0.027 μg/ml. Media was replenishedon days 4, 7 and 9. The co-culture model was maintained in either TCSOptimised medium (supplied with the co-culture assay) or in MCDB131medium supplemented with 2% foetal calf serum (FCS), 1% glutamine and 1%penicillin/streptomycin (hereafter referred to as 2% FS MCDB131 medium).The co-culture model was maintained at 37° C. in a humidified 5% CO₂/95%air atmosphere. Tubule formation was examined at day 11 following fixingand staining of tubules for CD31 using a tubule staining kit accordingto the manufacturers instructions (TCS Cell Works catalog #ZHA-1225).Briefly, cells were fixed with ice-cold 70% ethanol for 30 minutes atroom temperature (RT). Cells were blocked after which they were treatedwith anti-human CD31 for 60 minutes at RT. Plates were washed andtreated with goat anti-mouse IgG conjugated with alkaline phosphatase(AP) for 60 minutes at RT. After incubation with the AP-conjugatedsecondary antibody, the plates were washed and5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)substrate was added for approximately 10 minutes. The development of adark purple colour within 10 minutes reflected tubule formation. Plateswere subsequently washed and left to air dry. Quantification of tubulegrowth was conducted by whole-well image analysis methodology using aZeiss KS400 3.0 Image Analyser. The morphological parameter measured inthe quantification methodology was total tubule length. In someexperiments, the number of bifurcations in the tubes was also assessed.All tubule formations within each of the 24 wells were measuredexcluding a rim of 100 μm depth to avoid edge retraction artifact.

As illustrated in FIG. 2, it was observed that the antibodies areeffective in inhibiting endothelial cell tube formation in vitro.Furthermore, the potency of several of the antibodies in this assay wasdetermined. These data are summarized in Table 19. Taken together, thedata indicate that the antibodies are active in a functional assay thatmodels the angiogenic process.

TABLE 19 IC₅₀ (μg/ml) Antibody Isotype Vessel length # bifurcations 4B4IgG4 0.244 0.247 IgG1 0.135 0.152 21H3 IgG4 0.201 0.0740 IgG1 0.04680.0727 21H3RK IgG1 0.0507 0.0921 9G8 IgG4 0.656 0.363 2H10 IgG4 0.5140.446

Example 10 Determination of Binding Affinity of Purified Antibodies

The binding affinities of the purified antibodies for DLL4 was estimatedusing is both FACS and BIAcore techniques. For FACS affinitydetermination, HEK293 cells overexpressing either human or cynomolgusmonkey DLL4 were seeded at approximately 85,000-104,000 cells/well andincubated with titrations of purified antibody for 5 hours at 4° C. Thecells were then washed and incubated with goat anti-human IgG-Fc-Cy5+5μg/mL 7-Amino-Actinomycin (7AAD) for 30 minutes at 4° C. Bound DLL4 wasdetected using FACS analysis and data was fitted to the followingequation (see Drake & Klakamp, 2007, J. Immunol. Methods, 318, 157-162for derivation):

$F = {{P^{\prime}\frac{\left( {K_{D} + L_{T} + {n \cdot M}} \right) - \sqrt{\left( {K_{D} + L_{T} + {n \cdot M}} \right)^{2} - {4{n \cdot M \cdot L_{T}}}}}{2}} + B}$

In this equation, F=mean fluorescence, L_(T)=total molecular mAbconcentration, P′=proportionality constant that relates arbitraryfluorescence units to bound mAb, M=cellular concentration in molarity,n=number of receptors per cell, B=background signal, andK_(D)=equilibrium dissociation constant. For each mAb titration curve anestimate for K_(D) is obtained as P′, n, B, and K_(D) are allowed tofloat freely in the nonlinear analysis. Table 20 summarizes the affinityestimates for the anti-DLL4 antibodies for human and cynomolgus monkeyDLL4 derived using the methodology described above

For Biacore analysis, each purified anti-DLL4 antibody was immobilizedon a CM5 sensor chip within a T100 using standard amine coupling.Immobilization levels were kept below 200 RU. The concentration of DLL4was determined by UV-VIS spectroscopy using a molar absorptivity at 280nm of 110, 440 M⁻¹cm⁻¹, which was is calculated from the sequence of theprotein using a method developed by Pace et al. (G. R. Grimsley and C.N. Pace (2003) in Current Protocols in Protein Science (John Wiley &Sons, Inc.), 3.1.1-3.1.9). The antigen DLL4 (R&D systems; human catalog#1056-D4-050 or mouse, catalog #1389-D4-050) was diluted to a startingconcentration of 32-64 nM and tested in a 3-fold dilution series intriplicate. The running buffer contained HBS-P with 0.1 mg/ml BSA andbinding responses were collected at 23° C. Bound complexes wereregenerated with a 15 s pulse of 10 mM of sodium hydroxide. The responsedata were globally fitted with a simple 1:1 interaction model and Table20 summarizes the k_(a), k_(d) and K_(D) estimates obtained for theanti-DLL4 antibodies when binding to human soluble DLL4 was assessed. Inaddition, affinity estimates to soluble mouse DLL4 were also generatedfor 21H3, 21H3RK and 4B4 using BIAcore: all of the antibodies in IgGformat had an affinity of 360 nM or less for soluble mouse DLL4.

TABLE 20 Antibody Human FACS Cyno FACS ID Isotype K_(D) (pM) K_(D) (pM)21H3RK IgG1 155 66.7 21H3RK IgG2 225 N.T. 21H3 IgG1 103 78.1 21H3 IgG2320 N.T. 21H3 IgG4 359 N.T. 4B4 IgG1 157 81.0 4B4 IgG2 232 N.T. 4B4 IgG4516 N.T. 9G8 IgG4 999 N.T. 2H10 IgG4 882 N.T. N.T. = not tested.

TABLE 21 Antibody ID Isotype K_(a) (M⁻¹ s⁻¹) K_(d) (s⁻¹) K_(D) (pM)21H3RK IgG1 3.37 × 10⁵ 1.66 × 10⁻⁴ 493 21H3RK IgG2 2.22 × 10⁵ 1.60 ×10⁻⁴ 721 21H3 IgG1 2.88 × 10⁵ 1.71 × 10⁻⁴ 594 21H3 IgG2 2.59 × 10⁵ 1.55× 10⁻⁴ 598 21H3 IgG4 3.17 × 10⁵ 1.52 × 10⁻⁴ 481 4B4 IgG1 1.18 × 10⁵ 2.01× 10⁻⁵ 170 4B4 IgG2 1.07 × 10⁵ 3.60 × 10⁻⁵ 336 4B4 IgG4 1.18 × 10⁵ 3.33× 10⁻⁵ 283 9G8 IgG4 1.03 × 10⁵ 1.36 × 10⁻⁵ 132 2H10 IgG4 4.09 × 10⁴ 4.02× 10⁻⁵ 981

Example 11 Determination of Cross-Competition for Dll4 by FACS Analysis

The ability of the purified DLL4 antibodies to inhibit the binding ofother DLL4 antibodies to human DLL4 was assessed using a FACS assay.Briefly, antibodies were directly labelled with Alexa-647 using acommercially available labelling kit (e.g. Molecular Probes catalog#A30009, A-20186) as per the manufacturer's instructions. To determinethe level of cross-competition, unlabelled anti-DLL4 antibodies (finalconcentration: 10-0.01 μg/ml) diluted in PBS containing 2% FCS wereadded to DLL4-expressing HEK 293 cells (as described in example 6; 50000cells/well diluted in PBS containing 2% FCS) and incubated for 1 h at 4°C. Subsequently, Alexa-647 labeled anti-DLL4 antibodies were added at afinal concentration of 0.1 μg/ml and plates were incubated for a further1 h at 4° C. prior to washing and reading on a FACSCalibur instrument.Data that summarizes the ability of unlabeled antibodies (10 μl, 0.1μg/ml) to compete with labeled 21H3RK for binding to human DLL4 isincluded in FIG. 3. Additional data that summarizes the ability ofunlabeled antibodies to compete with labeled 21H3RK and 4B4 for bindingto human DLL4 is included in Table 22.

In addition, the ability of the antibodies to detect recombinant DLL4(R&D systems, catalog #1506-D4-050/CF) and native DLL4 expressed inHEK293 cells (see example 3) was also determined via Western blot usingstandard techniques. Briefly, DLL4 expressing HEK293 cells wereharvested in RIPA buffer (Thermo, catalog #89900 containing 1 proteaseinhibitor cocktail tablet (Roche, catalog #11873580001) and is proteinquantified using a BCA protein assay (Pierce, catalog #23227) accordingto the manufacturer's instructions. For Western blotting, proteinsamples were thawed on ice and incubated at 100° C. for 2 min prior tothe addition of 10× Nupage sample reducing agent (Invitrogen, catalog#NP0004) and 4× sample buffer prior to loading into pre-cast 4-12%NuPage BisTris gels (Invitrogen, catalog #NP0321B0X) in MES buffer(Invitrogen, catalog #NP0002). Following electrophoretic transfer tonitrocellulose, blots were blocked for 1 h in Tris-buffered saline (100mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.05% Tween 20 (TBST)containing 5% milk followed by incubation with either 9G8, 2H10, 21H3,4B4 or 20G8 (all 2 μg/ml) or commercially available anti-DLL4 antibodies(R&D Systems, catalog #MAB1389; Abcam, catalog #ab7280, both at 1 μg/ml)in TBST containing 5% milk. After incubation on an orbital shakerovernight at 4° C., blots were washed with TBST and incubated witheither anti-rat, anti-rabbit or anti-human-HRP-conjugated secondaryantibodies (Jackson Immunochemicals, catalog #112-035-003 and111-035-003, 1:20,000 dilution or KPL, catalog #074-1006, 1:10,000dilution) TBST containing 5% milk for 1 h at RT. Excess antibody wasremoved by washing as above and immunocomplexes were visualized viaenhanced chemiluminescence detection according to the manufacturer'sinstructions (Pierce, catalog #34076) and detected on Hyperfilm ECL(Amersham, catalog #28906839) or Biomax MR (Kodak, catalog #8952855)film. The results demonstrate that under these conditions, bothrecombinant and native DLL4 can be detected by the commercial antibodiesand 9G8/2H10, but not by 21H3/20G8 or 4B4, suggesting that theseantibodies may interact with different epitopes on DLL4 (data notshown).

TABLE 22 Competing antibody IC₅₀ (μg/ml) 21H3RK 21H3 4B4 9G8 2H10Labeled 4B4 0.223 0.274 0.348 0.856 1.15 antibody 21H3RK 0.214 0.2480.379 0.596 0.723

Example 12 Sequence Modifications to 9G8 and 2H10

Immunoglobulin genes undergo various modifications during maturation ofthe immune response, including recombination between V, D and J genesegments, isotype switching, and hypermutation in the variable regions.Recombination and somatic hypermutation are the foundation forgeneration of antibody diversity and affinity maturation, but they canalso generate sequence liabilities that may make commercial productionof such immunoglobulins as therapeutic agents difficult, or increase theimmunogenicity risk of the antibody. In general, mutations in CDRregions are likely to contribute to improved affinity and function,while mutations in framework regions may increase the risk ofimmunogenicity. This risk can be reduced by reverting frameworkmutations to germline, while ensuring that activity of the antibody isnot adversely impacted. Some structural liabilities may be generated bythe diversification processes, or they may exist within germlinesequences contributing to the heavy and light chain variable domains.Regardless of the source, it may be desirable to remove potentialstructural liabilities that may result in instability, aggregation,heterogeneity of product, or increased immunogenicity. Examples ofundesirable liabilities include unpaired cysteines (which may lead todisulfide bond scrambling, or variable sulfhydryl adduct formation),N-linked glycosylation sites (resulting in heterogeneity of structureand activity), as well as deamidation (e.g. NG, NS), isomerization (DG),oxidation (exposed methionine), and hydrolysis (DP) sites. In an effortto reduce the risk of immunogenicity, and improve pharmaceuticalproperties of lead antibodies, certain variants were generated andtested for binding and activity. Site directed mutagenesis was carriedout using the Stratagene Quick Change II kit, as described by themanufacturer. Variant sequences were expressed in the InVitrogenFreestyle system by transient transfection (following manufacturerrecommended protocols), and purified by Protein A affinitychromatography.

The activity of the mutated antibodies was assessed in two ways:firstly, the ability of the antibodies to block the binding of solubleNotch1/Fc to full length human DLL4 stably expressed in HEK293 cells asdescribed in Example 3 was determined and, secondly, the binding of theantibodies to the same cell line used in the receptor-ligand competitionstudies was determined. For the binding studies, HEK293 cells stablyoverexpressing human DLL4 were resuspended in PBS containing 2% FCS at ais concentration of 50,000 cells/well and incubated with titrations ofantibody (final concentrations 0.01-10 μg/ml) for 1 h at 4° C. The cellswere then washed and incubated with 0.31 μg/ml anti-human IgG-Fc-FITC(BD Pharmingen, cat#555786) for 30 minutes at 4° C. Bound DLL4 wasdetected using FACS analysis. Results of these studies are summarized inTable 23, which show the effects of specific mutations to sequences of9G8 and 2H10 on the ability of the antibodies to compete with Notch1 forbinding to human DLL4 or to bind to human DLL4 relative to unmutatedantibodies.

TABLE 23 RL comp Activity Binding Activity Clone Variant (IC₅₀ μg/ml)(EC₅₀ μg/ml) 9G8 IgG2 VH wt/VL wt 0.170 0.071 VH L38W/VL wt 0.133 0.070VH wt/VL S7P 0.181 0.082 VH wt/VL C33S 0.136 0.067 VH wt/VL C33A 0.302N.T. VH wt/VL C33G 0.358 N.T. VH wt/VL C33D 0.345 N.T. VH wt/VL C33R0.201 N.T. VH wt/VL C33Y 0.320 N.T. VH wt/VL V79A 0.211 0.095 VH L38W/VLS7P 0.155 0.074 VH L38W/VL C33S 0.107 0.051 VH L38W/VL V98A 0.240 N.T.2H10 IgG2 VH wt/VL wt 0.224 0.043 VH M93V/VL wt 0.264 N.T. VH wt/VL C33S0.084 0.031 VH wt/VL C33A 0.174 N.T. VH wt/VL C33G 0.314 N.T. VH wt/VLC33D 0.178 N.T. VH wt/VL C33R 2.97 N.T. VH wt/VL C33Y >3.00 N.T. VHM93V/VL C33S 0.138 N.T.

Example 13 Evaluation of the Antiangiogenic Efficacy in a Spheroid-Basedin Vivo Angiogenesis Assay

Human umbilical vein endothelial cell (HUVEC) spheroids were prepared asdescribed earlier (Korff and Augustin: J. Cell. Biol. 143: 1341-52,1998) by pipetting 100 endothelial cells (EC) in a hanging drop onplastic dishes to allow overnight spheroid formation. The following day,using the method previously described (Alajati et al., Nature Methods5:439-445, 2008), EC spheroids were harvested and mixed in aMatrigel/fibrin solution with single HUVECs to reach a final number of100,000 ECs as spheroids and 200,000 single ECs per injected plug.VEGF-A and FGF-2 were added at a final concentration of 1000 ng/ml. MaleSCID mice (5-8 weeks old) were subcutaneously injected with 500 μl ofthe cell/matrix suspension. The following day (day 1) treatmentcommenced. At day 21 the study was terminated. The matrix plugs wereremoved and fixed in 4% PFA. All matrix plugs were paraffin embedded andcut to a thickness of 8-10 μm for histological examination. Bloodvessels were visualized by staining for human CD34 and smooth muscleactin (SMA) and the vessel density and pericyte coverage was determined.Both IgG1 and IgG2 antibodies are effective in modulating vesselformation and inhibiting pericyte recruitment in vivo. The data obtainedsuggest that treatment with anti-DLL4 antibodies (21H3RK or 4B4) inducesan increase in human vessel formation of at least 100% over untreatedcontrol at antibody concentrations as low as 1 mg/kg. In addition, theseincreases in human vessel formation were associated with a decrease inpericyte coverage (as assessed by the percentage of human CD34 positivevessels that were also associated with cells positive for αSMAexpression) of at least 50% at antibody doses of 5 mg/kg. Datasummarizing the effect of 21H3RK IgG1 dosed twice weekly i.p. at 1, 0.2and 0.04 mg/kg is shown in Table 24. Taken together, the data indicatethat the antibodies are active in an in vivo assay of angiogenesis.

Number of Vessel coverage Treatment (2 × CD34+ve vessels(CD34+ve/αSMA+ve vessels) weekly) Mean s.e.m. Mean s.e.m. Vehicle 23537.3 49.9 2.64 21H3RK (1 645 78.6 15.9 2.38 mg/kg) 21H3RK (0.2 587 74.717.2 2.8 mg/kg) 21H3RK (0.04 478 61.8 27.1 3.66 mg/kg)

Example 14 Epitope Mapping of 21H3RK

Monoclonal 21H3RK binds specifically to human DLL4 but does notrecognize human DLL1. This specificity is employed to deduce the bindingepitope of Mab 21H3RK to human DLL4. Chimeric variants were engineeredwith portions of the extracellular domain of DLL4 replaced with thecorresponding segments of DLL1. Human DLL4 (Yoneya et al., 2001, J.Biochem., 129, 27-34, cloned in-house) and human DLL1 (accession #NM_(—)005618, Origene, MD) were used as templates in overlappingextension PCR to construct a series of variants which include the DLL4transmembrane domain for surface expression of the recombinant proteins.The resulting variants were cloned into a mammalian expression vectorencoding a human cytomegalovirus major immediate early (hCMVie)enhancer, promoter and 5′-untranslated region for transient mammalianexpression. The chimeric variants were transiently expressed in HEK293Fcells as membrane-bound proteins for flow cytometric characterizationwith Mab 21H3RK. 48 h post-transfection, HEK293F transfectants wereincubated with 1 μg/ml of Mab 21H3RK for 1 h on ice in PBS, washed, thenincubated with goat anti-human IgG-FITC (Jackson ImmunoResearchLaboratories, PA) and analyzed with a LSRII flow cytometer (BDBiosciences, CA). The expression levels of all chimeric variants were ismonitored by incubating with a mixture of both goat anti-mouse DLL4(which also recognizes human DLL4) and goat anti-human DLL1 polyclonalantibodies (both from R&D Systems, MN), then detected with porcineanti-goat IgG-PE (Invitrogen, CA).

Although human Dll1 and Dll4 share 53% identity at the amino acid level,Mab 21H3RK binds specifically to Dll4 but does not recognize Dll1.Chimeric variants of human Dll4 encoding small portions of human Dll1were constructed to identify the region responsible for thisspecificity. Twelve chimeric knock-out variants were constructed bysubstituting subdomains of human Dll4 with the corresponding residues ofhuman Dll1. FIG. 4 illustrates how the extracellular portion of DLL4 wasdivided into structurally defined sub domains. The large amino-terminus(N-ter) of the mature DLL4 protein was divided into two smallersegments. KO variant N-ter 1 replaces the first 86 amino acids (AA) ofthe mature DLL4 protein with human DLL1, and KO variant N-ter 2 replacesamino acids 87-146 with human DLL1. Other knockout variants representedin the figure include the Dll1 substitution of: the entire N-terminaldomain (AA 1-146), the DSL domain (AA 147-191), the EGF1 domain (AA192-224), both the EGF1 and 2 domains (AA 192-255), both the EGF3 and 4domains (AA 256-333), and the four EGF5-8 domains (AA 334-503).Additionally, combined domain substitution variants were engineered:both the N-terminal and DSL domains (AA 1-191), the N-terminal plus theDSL and EGF1-2 domains (AA1-255), the DSL and EGF1-2 domains (AA147-255), and the DSL and EGF1 domains (AA 147-224). All recombinantproteins were expressed well on the cell surface as monitored withanti-Dll4 and Dll1 polyclonal antibodies (FIG. 5, upper panels of bothrows), however Mab 21H3RK did not recognize any of the constructscomprised of both DSL and EGF1 human Dll1 domains (FIG. 5, bottompanel). Additionally, the Dll1 construct encoded with Dll4 DSL and EGF1domains (knock-in mutants) conferred Mab 21H3RK binding to Dll1 (FIG.6). Therefore, the binding epitope of 21H3RK is localized within the DSLand EGF1 domains.

To further refine the binding epitope within this segment of the proteinand identify the critical residues responsible for the Mab 21H3RKbinding specificity, three additional variants were engineered. Three,fifteen amino acid segments within the DSL and/or the EGF1 domains ofDll4 were substituted with the corresponding Dll1 residues: is fragmentA (AA 187-201), fragment B (AA 200-214), and fragment C (AA 210-224)encode only a few amino acid substitutions where Dll1 and Dll4 sequencesare not conserved. Fragment A spans the last five amino acids of the DSLdomain, the four amino acid linker between the DSL and EGF1 domain, andsix amino acids of the EGF1 domain. Substituting these 15 amino acidswith Dll1 residues resulted in the loss of Mab 21H3RK binding. No effectwas observed when Dll4 residues in fragments B and C were replaced withDll1. These data identifies the 15 amino acids (AA 187-201) includingC-terminus of DSL and N-terminus of EGF1 are important for binding, theepitope to 21H3RK has been mapped to DSL and EGF1 domains (AA 147-224)with the critical region localizing at C-terminus of DSL and N-terminusof EGF1 (AA187-201).

Example 15 Determination of Dll4 Antibodies to Cause Internalization ofDLL4 by FACS Analysis

The ability of the purified antibodies to induce internalization of DLL4was investigated by FACS analysis. HEK293 cells stably overexpressingDLL4 were dissociated and washed in FACS buffer (PBS+2% FCS) prior toplating at 50,000-100,000 cells per well in V-bottom plates. Primaryantibodies (anti-DLL4 or appropriate isotype control) were diluted to afinal concentration of 10 μg/ml in warm 37° C. FACS buffer and added tothe cells for 30, 60, 120 or 240 min in a tissue culture incubator (37°C./5% CO₂). At the appropriate time point, cells were spun at 500 g in acentrifuge prechilled to 4° C. and washed in cold FACS buffer, prior toincubation with a FITC labelled anti-human IgG secondary antibody (1μg/ml, Jackson Labs, cat #109-096-098) for 10 min on ice. Afterincubation, cells were respun at 500 g in a pre-chilled centrifuge,washed with cold FACS buffer and fixed with 2% paraformaldehye for 20min. Internalization was assessed by reading on a FACSCalibur. Underthese assay condiditions, <10% internalization of 21H3RK occurred at theabove time points. Internalization can also be determined by incubatingwith a non-cross-competing antibody to DLL4 instead of a secondaryanti-human IgG antibody. In some experiments, primary antibody (10μg/ml) diluted in FACS buffer was pre-incubated with DLL4 overexpressingcells as described above for 30 min on ice prior to washing in warm 37°C. FACS buffer and incubating cells in a tissue culture incubator (37°C./5% CO₂) for 30, 60, 120 or 240 min. After these incubations, cellswere washed and incubated with secondary antibody and fixed as describedabove prior to reading on a FACSCalibur. Under these conditions, <15%and <35% internalization relative to t=0 control was observed for 21H3RKand 4B4 at t=60 and 240 min, respectively. Internalization under theabove assay conditions can also be determined by incubating with anon-cross-competing antibody to DLL4 instead of a secondary anti-humanIgG antibody

Example 16 Activity of Anti-DLL4 Antibodies in a Mouse Matrigel PlugModel of Angiogenesis

The ability of anti-DLL4 antibodies to modulate angiogenesis can beassessed using a Matrigel plug assay. In this assay,angiogenesis-inducing compounds such as bFGF, VEGF or tumor cells can beintroduced into liquid Matrigel which, after subcutaneous injection,solidifies and permits infiltration by endothelial and vascular smoothmuscle cells and allows the formation of new blood vessels. Briefly,Matrigel in liquid form at 4° C. can be mixed with vehicle (e.g. PBS) orgrowth factors/tumor cells (e.g. LL2, MCF7, A431, Colo205, KNRK, Calu-6,SW620, Panc1) and 0.5 ml injected subcutaneously into the lowerabdominal area of female 129s1/SvlmJ mice (6-8 weeks old, n=5 pergroup). Anti-DLL4 antibodies can be administered twice weekly viaintraperitoneal injection. After 5-10 days, animals can be euthanizedhumanely and plugs can be recovered for assessment of angiogenesis,which can be determined by histological scoring of vessel density andmural cell coverage by for example, assessment of CD31 and alpha smoothmuscle actin (αSMA) immunostaining, measurement of haemoglobin content,and measurement of vessel perfusion using, for example, FITC-Dextran.The ability of anti-DLL4 antibodies to modulate angiogenesis is thusdetermined.

Example 17 Activity of Anti-DLL4 Antibodies in Human Tumor XenograftModels from Primary Patient Tumor Samples

This example describes the use of anti-DLL4 antibodies to inhibit orprevent the growth of tumors derived from primary patient samples whengrown as xenografts in mice. Briefly, the recipient mouse can beanesthetized by isoflurane inhalation until it has reached a surgicallevel of anesthesia. The primary tumor can then be rinsed with RPMIsupplemented with antibiotics and 10% FCSi before being minced toproduce a “slushy mixture” with scalpels and divided into appropriatevolumes for implantation (e.g., a 300 mg tumor can be implanted into 4mice). Tumor mixtures can be loaded into 13-gauge cancer implanttrocars. The shaft of the trocars can be completely filled with tumormixtures and inserted subcutaneously into the right flank and thecontents dispensed under the dorsal fat pad. The mouse can then bereturned to its cage and monitored for recovery.

In the first passage, usually 3-5 mice are implanted with primary tumormixture. When the tumors reach 800-1000 mm³, they are sliced intoapproximately 3×3×3 mm fragments and subpassaged into 5 mice with 1fragment into each mouse. The remaining tumor material is archived inRecovery™ Cell Culture Freezing medium (Gibco, catalog #12648-010) inaddition to H&E staining and DNA/RNA extraction. Tumors beyond passage 2can be used for implant for efficacy studies. In efficacy experiments, 1tumor fragment is implanted into each animal.

Tumor growth is followed by measuring 2 perpendicular diameters. Tumormeasurements and body weights can be recorded twice a week for 2 weeksafter the initiation of treatment. The formula for tumor volumecalculation is as following: (L×W²)/2.

DLL4 antagonistic antibodies can be dosed as a solution. Treatments canbe initiated when the average tumor volume reached approximately 100-200mm³ or at the same time as tumor implantation. The treatment period canconsist of a total of 28 days. DLL4 antagonistic antibodies can beadministered at for example, 5, 10 or 20 mg/kg/day (ip, qd, 2×/wk) as asingle agent or in combination with other agents. Tumor measurements andbody weights are recorded twice a week for 4 weeks after the initiationof treatment. The ability of DLL4 antibodies to inhibit the growth oftumor xenografts derived from patient samples either alone or incombination is thus determined.

What is claimed is: 1-19. (canceled)
 20. An isolated nucleic acidmolecule encoding an antibody or binding fragment thereof thatspecifically binds to DLL4, wherein the antibody comprises: (a) a heavychain variable region (VH) CDR1, CDR2 and CDR3 of SEQ ID NO:30; and (b)a light chain variable region (VL) CDR1, CDR2 and CDR3 of SEQ ID NO:32.21-24. (canceled)
 25. The nucleic acid molecule according to claim 20wherein the antibody or binding fragment thereof comprises the VH aminoacid sequence as shown in SEQ ID NO:30 or SEQ ID NO:75.
 26. The nucleicacid molecule according to claim 20 wherein the antibody or bindingfragment thereof comprises the VL amino acid sequence as shown in SEQ IDNO:32 or SEQ ID NO:50 or SEQ ID NO:76.
 27. The nucleic acid moleculeaccording to claim 25 wherein the antibody or binding fragment thereofcomprises the VH amino acid sequence as shown in SEQ ID NO:30.
 28. Thenucleic acid molecule according to claim 26 wherein the antibody orbinding fragment thereof comprises the VL amino acid sequence as shownin SEQ ID NO:32.
 29. The nucleic acid molecule according to claim 26wherein the antibody or binding fragment thereof comprises the VL aminoacid sequence as shown in SEQ ID NO:50.
 30. The nucleic acid moleculeaccording to claim 27 wherein the antibody or binding fragment thereofcomprises the VL sequence as shown in SEQ ID NO:32.
 31. The nucleic acidmolecule according to claim 27 wherein the antibody or binding fragmentthereof comprises the VL sequence as shown in SEQ ID NO:50.
 32. Thenucleic acid molecule according to claim 20 wherein the VH is encoded bythe nucleotide sequence as shown in SEQ ID NO:29 and the VL is encodedby the nucleotide sequence as shown in SEQ ID NO:49.
 33. The nucleicacid molecule according to claim 20 wherein the VH is encoded by thenucleotide sequence as shown in SEQ ID NO:29 and the VL is encoded bythe nucleotide sequence as shown in SEQ ID NO:31.